U.S. patent application number 15/796755 was filed with the patent office on 2018-06-07 for protein-protein interaction as biomarkers.
The applicant listed for this patent is H. LEE MOFFITT CANCER CENTER AND RESEARCH INSTITUTE, INC.. Invention is credited to ERIC B. HAURA.
Application Number | 20180156798 15/796755 |
Document ID | / |
Family ID | 47996745 |
Filed Date | 2018-06-07 |
United States Patent
Application |
20180156798 |
Kind Code |
A1 |
HAURA; ERIC B. |
June 7, 2018 |
PROTEIN-PROTEIN INTERACTION AS BIOMARKERS
Abstract
The subject invention pertains to materials and methods for the
classification of cancers as sensitive or resistant to treatments
based on protein-protein interactions, treatment of cancer,
identification of biomarkers, identification of protein-protein
interaction modulators, and selection of cancer treatments.
Inventors: |
HAURA; ERIC B.; (TAMPA,
FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
H. LEE MOFFITT CANCER CENTER AND RESEARCH INSTITUTE, INC. |
Tampa |
FL |
US |
|
|
Family ID: |
47996745 |
Appl. No.: |
15/796755 |
Filed: |
October 28, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14348152 |
Mar 28, 2014 |
9804160 |
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PCT/US2012/057633 |
Sep 27, 2012 |
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15796755 |
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61540212 |
Sep 28, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/57484 20130101;
G01N 2800/52 20130101; G01N 33/56966 20130101; G01N 33/57407
20130101 |
International
Class: |
G01N 33/569 20060101
G01N033/569; G01N 33/574 20060101 G01N033/574 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
No. CA119997 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method for assessing the sensitivity of a malignancy to a
potential treatment, comprising comparing a protein-protein
interaction (PPI) profile obtained from a sample of the malignancy
to a reference PPI profile.
2. The method of claim 1, wherein the sample PPI profile represents
the abundance of target binding partners that are in proximity to
each other within the sample.
3. The method of claim 2, wherein the sample PPI profile represents
the abundance of two binding partners that are in proximity to each
other within the sample.
4. The method of claim 2, wherein the sample PPI profile represents
the abundance of three or more binding partners that are in
proximity to each other within the sample.
5. The method of claim 1, wherein the sample PPI profile represents
the abundance of target binding partners that are in proximity to
each other within the sample, and wherein at least one of the
target binding partners is a receptor tyrosine kinase (RTK).
6. The method of claim 1, wherein at least one of the target
binding partners is of an RTK class selected from among RTK class I
(EGF receptor family; ErbB family), RTK class II (insulin receptor
family), RTK class III (PDGF receptor family), RTK class IV (FGF
receptor family) RTK class V (VEGF receptors family), RTK class VI
(HGF receptor family), RTK class VII (Trk receptor family), RTK
class VIII (Eph receptor family), RTK class IX (AXL receptor
family), RTK class X (LTK receptor family), RTK class XI (TIE
receptor family), RTK class XII (ROR receptor family), XIII (DDR
receptor family), RTK class XIV (RET receptor family), RTK class XV
(KLG receptor family), RTK class XVI (RYK receptor family), and RTK
class XVII (MuSK receptor family).
7. The method of claim 2, wherein the binding partners of the PPI
and the downstream effectors of the PPI do not harbor any sequence
mutations known to be associated with sensitivity to a treatment
such as a PPI modulator.
8. The method of claim 2, wherein the sample PPI profile and the
reference PPI profile are each expressed as a value representative
of the abundance of target binding partners in proximity to each
other within the sample.
9. A method for treating a malignancy in a subject, comprising
administering a protein-protein interaction (PPI) modulator to the
subject, wherein the subject is predetermined to be sensitive to
the PPI modulator based on a PPI profile obtained from a sample of
the malignancy.
10. A method for treating a malignancy in a subject, comprising:
(a) assessing the sensitivity of a malignancy in the subject,
comprising comparing a protein-protein interaction (PPI) profile
obtained from a sample of the malignancy to a reference PPI
profile; and (b) administering a PPI modulator to the subject if
the malignancy is assessed to be sensitive to the PPI modulator;
and withholding the PPI modulator from the subject if the
malignancy is assessed to be resistant to the PPI modulator.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 14/348,152, filed Mar. 28, 2014, now U.S. Pat.
No. 9,804,160, which is the National Stage of International
Application No. PCT/US2012/057633, filed Sep. 27, 2012, which
claims the benefit of U.S. Provisional Application Ser. No.
61/540,212, filed Sep. 28, 2011, each of which is hereby
incorporated by reference herein in its entirety, including any
figures, tables, nucleic acid sequences, amino acid sequences, and
drawings.
BACKGROUND OF THE INVENTION
[0003] A better understanding of tumor subsets that benefit from
treatment is of critical importance to enable personalized
medicine. For this reason, a number of molecular approaches to
classify cancer and identify responsive subsets are now being
tested. Proteomic strategies (which examine global patterns of
protein expression or phosphorylation) are also being used to
identify subsets of tumors. This includes classic
immunohistochemistry approaches to measure protein expression in
paraffin fixed tumor sections, use of phospho-specific antibodies
to measure specific phosphorylation events on particular proteins,
proteomic profiling tools (such as reverse phase protein arrays),
and mass spectrometry based approaches. Biomarker systems to
measure protein-protein interaction biomarkers in cancer have
lagged behind these other tools. This is an important missing
component of most biomarker strategies, as cellular signaling
requires proper formation of signaling complexes and networks of
proteins that act in concert to produce a physiological signal.
[0004] It is known that oncoproteins, such as epidermal growth
factor receptor (EGFR) or V-Ki-ras2 Kirsten rat sarcoma viral
oncogene homolog (KRAS), produce a signaling network comprised of a
defined set of molecules that lead to "oncogene addiction" and cell
transformation. A better understanding of the oncoprotein signaling
network could uncover novel therapeutic targets or therapeutic
strategies and allow "network medicine" to become a reality.
BRIEF SUMMARY OF THE INVENTION
[0005] The subject invention pertains to materials and methods for
the identification of malignancies as suitable for treatment,
treatment of malignancies, and selection of treatments
(prophylactic and/or therapeutic interventions). The oncoprotein
signaling network can provide biomarkers categorized by
protein-protein interactions (PPI) that carry information to guide
clinical decision making. The present invention includes materials
and methods for determining the responsiveness of a malignancy to a
therapeutic intervention, such as a modulator (inhibitor or
inducer) of the PPI, by measuring PPI in a sample of the
cancer.
[0006] Aspects of the invention include a method for assessing the
sensitivity of a malignancy to a potential treatment; a method for
treating a malignancy in a subject that is predetermined to be
sensitive to a PPI modulator based on a PPI profile obtained from a
sample of the malignancy; a kit for quantitatively detecting the
proximity of target binding partners within a cancer PPI network; a
kit for quantitatively detecting the proximity of target binding
partners within a tertiary interaction (an interaction of three or
more target binding partners); a method for the identification of a
biomarker; and a method for identifying an agent as a PPI
modulator.
[0007] Assays such as proximity ligation assays (PLAs) can be used
to quantitatively measure defined PPI in the cancer sample and
determine PPI expression patterns to establish a relationship to
clinical outcome. PLA technology is capable of detecting single
protein events such as protein interactions (e.g., protein
dimerization) and modifications (e.g., protein phosphorylation) in
tissue and cell samples prepared for microscopy. Two interacting
proteins in a complex are identified with two primary antibodies
(of different species) specific for the protein--one primary
antibody for each interacting protein (Soderberg O. et al., "Direct
observation of individual endogenous protein complexes in situ by
proximity ligation", Nat. Methods., 2006 December; 3(12):995-1000;
and Jarvius M. et al., "In situ detection of phosphorylated
platelet-derived growth factor receptor beta using a generalized
proximity ligation method", Mol. Cell Proteomics, 2007 September;
6(9):1500-9, which are each incorporated herein by reference in its
entirety"). Species-specific secondary antibodies (referred to
herein as "PLA probes" or "proximity probes") each have a unique
short nucleic acid strand attached to it. When the PLA probes are
in close proximity (e.g., less than 40 nm), the nucleic acid
strands (through the addition of two circle-forming
oligonucleotides) become ligated together by enzymatic ligation and
form a circle that functions as a template for amplification (see
FIG. 2A). Because the distance of the nucleic acid strands is
small, only proteins that interact will allow ligation. After the
amplification reaction, the resulting rolling circular
amplification (RCA) serves as a target for labeled (e.g.,
fluorescently labeled) complimentary oligonucleotide probes (see
FIG. 2B), which allow for detection (e.g., visualization) and
quantification of the product (e.g., using a florescent
microscope). This technique greatly amplifies the signal for each
antigen recognition event. In this way, the signal is amplified by
generating an amplified nucleic acid surrogate of the binding
partners (proteins) in sufficient proximity to produce a signal.
The signal from each detected pair of proximity probes can be
visualized, e.g., as a fluorescent dot, and quantified (counted)
and assigned to a specific subcellular location (localization of
signal) based on microscopy images.
[0008] The methods and kits of the invention make use of PLAs to
measure PPI in cell samples, e.g., cancer samples. The PPIs may be
binary (having two protein binding partners) or tertiary (having
three or more protein binding partners). Proteins within a tertiary
interaction may be bound to one or more proteins within the PPI.
For measurement of binary interactions, oligonucleotide probes for
one type of signal (for example, one color, such as red). For
measurement of tertiary interactions, a second signal that is
discernable from the first signal is required for detection of the
second interaction. Instead of two primary antibodies, three
primary antibodies can be used, each of a different species (mouse,
rabbit, and goat, for example). For example, in measuring a
tertiary interaction between protein binding partners represented
as A, B, and C, two separate PLAs are carried out, with a first
signal (e.g., red signal) representing interaction between A and B,
and a second signal (distinguishable from the first signal)
representing interaction between B and C (e.g., green signal).
Optionally, for visualization, the images can be constructed
showing each signal (e.g., red and green signals) with overlay.
[0009] PPI measurements can be taken of virtually any oncoprotein
and its binding partner(s). In some embodiments, the oncoprotein
comprises epidermal growth factor receptor (EGFR). As EGFR is a key
kinase in lung cancer, in some embodiments, the oncoprotein
comprises EGFR and the cancer sample is a lung cancer sample.
[0010] One aspect of the invention concerns a method for assessing
the sensitivity of a malignancy to a treatment based on PPI. The
method for assessing the sensitivity of a malignancy to a treatment
comprises comparing a protein-protein interaction (PPI) profile
obtained from a sample of the malignancy to a reference PPI
profile. The sample PPI profile represents the abundance of target
binding partners that are in proximity to each other within the
sample. A negative result (lack of PPI) in a sample would indicate
a lack of sensitivity to the PPI modulator and be useful clinically
to avoid giving patients unnecessary PPI modulator therapy. A
positive result (presence of PPI) would indicate that the
malignancy is potentially sensitive to the PPI modulator and allow
the clinician to give the PPI modulator therapy to those patients
who would be most likely to benefit. As a specific example, the
method may be used for determining whether there is no EGF
signaling in a sample such as spleen tissue, and this kind of a
negative result can be useful clinically to avoid giving patients
unnecessary EGF receptor inhibitor therapy, or alternatively be
used to give the therapy to those patients who would be most likely
to benefit.
[0011] PPI profiles (e.g., sample PPI profile, and reference PPI
profile) may each be expressed as a value representative of the
abundance of target binding partners in proximity to each other
within the sample. The sample PPI profile and reference PPI profile
may be expressed by any method useful for comparison purposes, such
as a numeric value, score, cutoff (threshold), or other expression.
For example, a negative result in which a PPI profile in a sample
does not reach the cutoff would be useful clinically to avoid
giving patients unnecessary PPI modulator therapy. A positive
result in which a PPI profile in a sample is at or above the cutoff
would indicate potential sensitivity and allow the clinician to
give the PPI modulator therapy to those patients who would be most
likely to benefit.
[0012] In some embodiments of the methods and kits of the
invention, the target PPI is that of a known cancer signaling
network. Binding members of a target PPI may include protein
products of oncogenes or tumor suppressor genes, for example. In
some embodiments, the sample PPI profile represents the abundance
of target binding partners that are in proximity to each other
within the sample, wherein at least one of the target binding
partners is selected from among EGFR, ALK, MET, IGFR, Src, ErbB3,
Mig6, Grb2, Sts1, p85, and Hsp90.
[0013] In some embodiments of the methods and kits of the
invention, the target binding partners are selected from among EGFR
and ErbB3; EGFR and Grb2; EGFR and Mig6; EGFR and Sts1, EGFR and
Src; EGFR and Hsp90; ErbB3 and p85; ALK and EML4; MET and Gab1;
IGFR and IRS; Hsp90 and Cdc37; ALK and Shc1; EGFR and Shc1.
[0014] In some embodiments of the methods and kits of the
invention, the PPI comprises a tyrosine kinase, such as the human
tyrosine kinases listed in Tables 3 and 4. In some embodiments, the
PPI comprises an interaction between the binding partners listed in
Table 5 (human tyrosine kinase interactions).
[0015] PLAs can be used to generate biomarkers against receptor
tyrosine kinases (RTKs). RTKs are important proteins in cancer and
highly "druggable" targets. Approximately twenty different RTK
classes have been identified thus far. In some embodiments of the
methods and kits of the invention, at least one of the target
binding partners is an RTK. In some embodiments of the methods and
kits of the invention, at least one of the target binding partners
is of an RTK class selected from among RTK class I (EGF receptor
family; ErbB family), RTK class II (insulin receptor family), RTK
class III (PDGF receptor family), RTK class IV (FGF receptor
family) RTK class V (VEGF receptors family), RTK class VI (HGF
receptor family), RTK class VII (Trk receptor family), RTK class
VIII (Eph receptor family), RTK class IX (AXL receptor family), RTK
class X (LTK receptor family), RTK class XI (TIE receptor family),
RTK class XII (ROR receptor family), XIII (DDR receptor family),
RTK class XIV (RET receptor family), RTK class XV (KLG receptor
family), RTK class XVI (RYK receptor family), and RTK class XVII
(MuSK receptor family).
[0016] In some embodiments of the methods and kits of the
invention, one or more of the binding partners of the target PPI
have one or more sequence mutations that are known to be associated
with occurrence of the malignancy. In other embodiments, the
binding partners of the target PPI do not harbor (lack) any
sequence mutations known to be associated with occurrence of the
malignancy, or with sensitivity to a treatment such as a PPI
modulator. In some embodiments, the binding partners of the target
PPI and the downstream effectors of the target PPI do not harbor
any sequence mutations known to be associated with occurrence of
the malignancy, or with sensitivity to a treatment such as a PPI
modulator. Such sequence aberrations in a subject or in a sample
can be detected using methods known in the art (e.g., mutation
analysis). Typically, abnormalities in nucleic acid sequences are
identified by comparison to reference sequence data (sequences of
normal cells or cancer cells) on databases, such as GenBank and
EMBL, and specific data resources such as Cancer Gene Census
(mutated genes causally implicated in human cancer), COSMIC
(Catalogue of Somatic Mutations in Cancer), and CGP Resequencing
Studies (somatic mutations from large scale resequencing of genes
in human cancer). Mutations causing or contributing to cancer may
be large-scale mutations, involving the deletion or addition of a
portion of a chromosome, or small-scale mutations, including point
mutations, deletions, insertions, which may occur in the promoter
region of a gene and affect its expression, may occur in the coding
sequence and alter the stability or function of the gene's protein
product.
[0017] The sample may be any cell sample potentially harboring the
target protein(s). For example, a cytology sample may be obtained
from a tissue selected from breast, ovaries, esophagus, stomach,
colon, rectum, anus, bile duct, brain, endometrium, lung, liver,
skin, prostate, kidney, nasopharynx, pancreas, head and neck,
kidney, lymphoma, leukemia, cervix, and bladder. The sample may be
a solid or non-solid tumor specimen. The tumor specimen may be a
carcinoma. The sample may be a new cancer, recurrent cancer,
primary cancer, or metastasized (secondary) cancer.
[0018] The sample may be obtained by methods known in the art, such
as surgery, biopsy, or from blood (e.g., circulating tumor cells),
ascites, or pleural effusion. The sample may be processed using
methods known in the art. For example, the sample may be fresh,
frozen, or formalin-fixed and paraffin-embedded (FFPE).
[0019] Preferably, the treatment against which the sample is being
assessed for sensistivity/resistance is a PPI modulator (i.e., a
PPI inhibitor or PPI inducer). However, the treatment may be a
treatment other than a PPI modulator.
[0020] Another aspect of the invention concerns a method for
treating a malignancy in a subject, comprising administering a
protein-protein interaction (PPI) modulator to the subject, wherein
the subject is predetermined to be sensitive to the PPI modulator
based on a PPI profile obtained from a sample of the malignancy. In
some embodiments, the PPI modulator is an inhibitor of the PPI of
the PPI profile (i.e., a PPI inhibitor). In some embodiments, the
PPI modulator is an inducer of the PPI of the PPI profile (i.e., a
PPI inducer).
[0021] Another aspect of the invention concerns a method for
treating a malignancy in a subject, comprising:
[0022] (a) assessing the sensitivity of a malignancy in the
subject, comprising comparing a protein-protein interaction (PPI)
profile obtained from a sample of the malignancy to a reference PPI
profile; and
[0023] (b) administering a PPI modulator to the subject if the
malignancy is assessed to be sensitive to the PPI modulator; and
withholding the PPI modulator from the subject if the malignancy is
assessed to be resistant to the PPI modulator. In some embodiments,
the PPI modulator is an inhibitor of the PPI of the PPI profile
(i.e., a PPI inhibitor). In some embodiments, the PPI modulator is
an inducer of the PPI of the PPI profile (i.e., a PPI inducer).
[0024] The invention includes also includes kits useful for
carrying out methods of the invention, (e.g., methods for the
classification of cancers as sensitive or resistant to treatments
based on protein-protein interactions, treatment of cancer,
identification of biomarkers, identification of protein-protein
interaction modulators, and selection of cancer treatments). Thus,
one aspect of the invention concerns a kit for detecting the
proximity of target binding partners within a cancer
protein-protein interaction network, comprising a primary antibody
to at least one of the target binding partners, and a proximity
probe comprising a secondary antibody (that binds to the primary
antibody) with an oligonucleotide conjugated thereto. In preferred
embodiments, the kit comprises:
[0025] a first primary antibody to a first target binding
partner;
[0026] a second primary antibody to a second target binding
partner;
[0027] a first proximity probe comprising a first secondary
antibody (that binds to the first primary antibody) with an
oligonucleotide conjugated thereto; and
[0028] a second proximity probe comprising a second secondary
antibody (that binds to the second primary antibody) with an
oligonucleotide conjugated thereto, wherein when the
oligonucleotides of the first and second proximity probes are in
sufficient proximity to each other, the oligonucleotides of the
proximity probes interact in the presence circle-forming
oligonucleotides by enzymatic ligation and form a circular product
that is amplified by rolling circle replication, producing an
amplification product. Optionally, the kit further comprises a
labeled oligonucleotide probe that hybridizes with the
amplification product, allowing detection and quantification of the
amplification product (representing the association (close
proximity) of the target binding partners).
[0029] Another aspect of the invention concerns a kit for detecting
the proximity of target binding partners within tertiary PPI (a
complex having three or more protein binding partners), referred to
herein as the "tertiary interaction kit". The target PPI may be
within a cancer signaling network, but is not limited to such
applications. The tertiary interaction kit can be used to study
viruses, for example, in which a viral protein forms an interaction
with protein A and protein B in a cell, giving rise to a tertiary
complex. Identification of this tertiary PPI may be diagnostic of
an active infection or yield important information about the
prognosis or predict the correct therapy. In cancer, a tertiary
complex may provide important information about the therapeutic
efficacy.
[0030] The tertiary interaction kit comprises:
[0031] a first primary antibody to a first target binding partner
of the tertiary interaction;
[0032] a second primary antibody to a second target binding partner
of the tertiary interaction;
[0033] a third primary antibody to a third target binding partner
of the tertiary interaction;
[0034] a first proximity probe comprising a first secondary
antibody (that binds to the first primary antibody) with an
oligonucleotide conjugated thereto; and
[0035] a second proximity probe comprising a second secondary
antibody (that binds to the second primary antibody) with an
oligonucleotide conjugated thereto;
[0036] a third proximity probe comprising a third secondary
antibody (that binds to the third primary antibody) with an
oligonucleotide conjugated thereto;
[0037] wherein when the oligonucleotides of the first and second
proximity probes are in sufficient proximity to each other, the
oligonucleotides of the first and second proximity probes interact
in the presence circle-forming oligonucleotides by enzymatic
ligation and form a circular product that is amplified by rolling
circle replication, producing a first amplification product;
and
[0038] wherein when the oligonucleotides of the second and third
proximity probes are in sufficient proximity to each other, the
oligonucleotides of the second and third proximity probes interact
in the presence of circle-forming oligonucleotides by enzymatic
ligation and form a circular product that is amplified by rolling
circle replication, producing a second amplification product.
Optionally, the kit detecting proximity of binding partners within
a tertiary PPI further comprises a first labeled oligonucleotide
probe that hybridizes with the first amplification product,
allowing detection and quantification of the first amplification
product (representing the association (close proximity) of the
first and second target binding partners), and a second labeled
oligonucleotide probe that hybridizes with the second amplification
product, allowing detection and quantification of the second
amplification product (representing the association of the second
and third target binding partners). Each primary antibody is of a
different species (e.g., mouse, rabbit, and goat, for example).
Preferably, the label of the first labeled oligonucleotide yields a
signal (e.g., a color) that is distinguishable from that of the
second labeled oligonucleotide (red and green, for example). An
additional primary antibody, proximity probe, and (optionally)
oligonucleotide probe can be included in the kit for each
additional target binding partner within the tertiary PPI to be
measured. Optionally, images can be constructed for visualization
showing each signal with overlay (e.g., red and green overlay).
[0039] In the various kits of the invention, each kit can include
instructions or packaging materials that describe how to use a
compound or composition (e.g., a reagent such as a primary
antibody, a secondary antibody, a labeled oligonucleotide probe
that hybridizes with the amplification product) of the kit. Within
the kit, the secondary antibody may be uncojugated or conjugated to
an oligonucleotide (making the secondary antibody a proximity
probe). The kits may also comprise, e.g., polymerase (for the
amplification reaction), ligase (for the ligation reaction), a
buffering agent, a preservative, or a protein stabilizing
agent.
[0040] The kits may also comprise components necessary for
detecting the label (e.g., an enzyme or substrate). The kit may
also contain a control sample or a series of control samples that
can be assayed and compared to a test sample. Each kit can include
one or more containers for individually enclosing each component of
the kit. Containers of the kits can be of any suitable material,
e.g., glass, plastic, metal, etc., and of any suitable size, shape,
or configuration. The one or more containers are can be enclosed
within outer packaging.
[0041] The kits of the invention may be used by contacting the
cell-containing sample with the primary antibodies (allowing the
primary antibodies to bind to their respective protein targets),
contacting the sample with the proximity probes (allowing the
proximity probes to bind to their respective antibody targets),
allowing the nucleic acid strands (also referred to as arms) of the
proximity probes to ligate if binding in close proximity (adding
ligase if necessary), amplifying the ligation product (adding
polymerase if necessary) to produce an amplification product, and
measuring the amplification product corresponding to the
interaction of each pair of protein targets within the PPI.
Measurement of the amplification product can be made using methods
known in the art for detecting and quantifying nucleic acid
amplification products, e.g., by adding a labeled oligonucleotide
that hybridizes to a sequence of the amplification product, and
analyzing the sample by visualizing the amplification product (as
represented by the label signal) with an appropriate visualization
device, such as a confocal or fluorescence microscope.
[0042] Another aspect of the invention concerns a method for
measuring protein-protein interactions having three or more binding
partners (a tertiary interaction) in a sample, comprising:
[0043] contacting the sample with three or more primary antibodies
to three or more corresponding target binding partners within a
target tertiary interaction;
[0044] contacting the sample with three or more proximity probes
(first, second, and third proximity probes) comprising a secondary
antibody that binds to the corresponding first antibody, wherein
each proximity probe has an oligonucleotide conjugated thereto;
[0045] wherein when the oligonucleotides of the first and second
proximity probes are in sufficient proximity to each other, the
oligonucleotides of the first and second proximity probes interact
in the presence circle-forming oligonucleotides by enzymatic
ligation and form a circular product that is amplified by rolling
circle replication, producing a first amplification product; and
[0046] wherein when the oligonucleotides of the second and third
proximity probes are in sufficient proximity to each other, the
oligonucleotides of the second and third proximity probes interact
in the presence of circle-forming oligonucleotides by enzymatic
ligation and form a circular product that is amplified by rolling
circle replication, producing a second amplification product; and
measuring the first and second amplification products.
[0047] In some embodiments, measurement of the first and second
amplification products comprises contacting the sample with two or
more labeled oligonucleotides, comprising a first labeled
oligonucleotide that hybridizes to a sequence of the first
amplification product, and a second labeled oligonucleotide that
hybridizes to a sequence of the second amplification product, to
produce labeled amplification products, wherein the labels are
distinguishable from one another; and measuring the PPIs by
visualizing the labeled amplification products. An additional
primary antibody, proximity probe, and labeled oligonucleotide can
be used for each additional target binding partner within the
tertiary PPI.
[0048] Another aspect of the invention concerns a method for the
identification of a biomarker, comprising selecting two or more
target binding partners within a cancer sample; generating a PPI
profile for the two or more target binding partners; and comparing
the PPI profile to the responsiveness of the cancer to a treatment
in vitro and/or in vivo (for example, in xenograft animal models or
human subjects). Correlation between the PPI profile to the
responsiveness of the cancer to a treatment of the cancer in vitro
and/or in vivo is indicative of a biomarker for treatment
responsiveness for the cancer. In some embodiments, the treatment
is a kinase inhibitor (e.g., a tyrosine kinase inhibitor (TKI)). In
some embodiments, at least one of the two or more target binding
partners comprises a receptor tyrosine kinase (RTK). In some
embodiments, RTK is within RTK class I-XVII. Preferably,
responsiveness of the treatment to the cancer in vitro, or in vivo
with xenograft animal models, is determined with the subject's
cancer cells; however, cancer cell lines known to be predictive of
responsiveness to the subject's cancer type may be utilized to
compare with the PPI profile.
[0049] Another aspect of the invention concerns a method for
identifying an agent as a PPI modulator, comprising: contacting
cancer cells with a candidate agent in vitro or in vivo; and
determining whether the candidate agent modulates a selected PPI in
a sample of the cancer cells. The candidate agent may be any
substance that potentially modulates (increases, decreases, or
otherwise alters) the PPI. The candidate agent may be a small
molecule, polypeptide, or nucleic acid, for example. Determination
of changes in PPI can be made by comparing a first PPI profile of
the cancer cells obtained prior to the contacting step to a second
PPI profile of the cancer cells obtained after the contacting step,
wherein a change in the PPI is indicative of a PPI modulator.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIGS. 1A-1D show physical mapping of protein-protein
interactions involving EGFR, showing 201 interactions with EGFR.
FIGS. 1A-1D demonstrate how interaction data (e.g., from literature
or affinity-purification mass spectrometry) can be used to generate
pairs of proteins to study as biomakers for treatment efficacy
using proximity ligation assays.
[0051] FIGS. 2A and 2B are diagrams showing the proximity ligation
assay (PLA) scheme.
[0052] FIGS. 3A-3F show results of an EGFR-GRB2 PLA in situ. Red
dots are signal of EGFR-GRb2 interaction detected by PLA using
confocal images with 10 z-sections merged. FIG. 3A shows strong
signals in PC9 cell line that are dramatically reduced after
treated with erlotinib (FIG. 3B). Negative controls are shown in
FIGS. 3C to 3F: no anti-rabbit 2.sup.nd antibody with PLUS DNA
chain (FIG. 3C); no anti-mouse 2.sup.nd antibody with MINUS DNA
chain (FIG. 3D), no rabbit anti-Grb2 antibody (FIG. 3E); no mouse
anti-EGFR antibody (FIG. 3F).
[0053] FIGS. 4A and 4B show results of EGFR-Grb2 PLA in the PC9
lung cancer cell line. PC9 lung cancer cells harboring an
activating EGFR mutation were examined for PLA using EGFR and Grb2
antibodies. Control untreated cells (FIG. 4A) were examined as well
as cells exposed to erlotinib, a small molecule epidermal growth
factor tyrosine kinase inhibitor (EGFR TKI) (FIG. 4B). Erlotinib
inhibits EGFR tyrosine phosphorylation and prevents Grb2 binding
(data now shown) thus serving as a perturbation to examine PLA. As
shown here, the EGFR-Grb2 PLA signal (red dots) is strong across
multiple cells and strongly repressed by erlotinib. Blue stain
=DAPI that stains nuclei. This figure also demonstrates the
feasibility of performing EGFR-Grb2 PLA (and potential other useful
PLA biomarkers) on low numbers of cells (and may work with cytology
specimens or other scant tumor biopsy specimens).
[0054] FIGS. 5A and 5B show results of EGFR-Grb2 PLA in lung cancer
tumor tissues (in vivo). Formalin-fixed paraffin-embedded (FFPE)
tumor tissue from a surgically resected adenocarcinoma of the lung
was used for EGFR-Grb2 PLA. As a negative control, FFPE spleen
tissue was chosen, as this tissue does not express EGFR protein. As
shown in FIG. 5A, the lung adenocarcinoma has strong signal (red
dots), compared to spleen that has no signal, shown in FIG. 5B. The
blue signal is DAPI that stains for nuclei.
[0055] FIGS. 6A and 6B show results of ALK-Grb2 PLA in H3122 cells.
PLAs have also been generated for detecting interactions between
ALK and Grb2. H3122 cells harboring an ELM4-ALK gene fusion were
examined using primary antibodies against the ALK receptor tyrosine
kinase and Grb2. The negative control consisted of the same assay
but leaving out the Grb2 antibody. In FIG. 6A, the small red foci
are identified, indicative of ALK-Grb2 interactions, while no foci
are observed in the negative contro (FIG. 6B). This indicates the
PLAs can be generated to identify tumors driven by other receptor
tyrosine kinases (beyond EGFR), including EML4-ALK transformed lung
cancers.
[0056] FIG. 7 shows results of examination of lung cancer cell
lines and spleen tissue for EGFR:Grb2 PLA signals related to EGFR
mutation status and drug sensitivity. This included cells harboring
EGFR mutation (HCC827, PC9, H4006, H1650). All these cells had high
levels of EGFR:Grb2 PLA signal. H1648, H322, H358, and H292 cells
also demonstrated intermediate to higher levels of PLA signal;
these cells have wildtype EGFR yet have some degree of sensitivity
to EGFR TKI (erlotinib). Finally, A549, H23, and H460 cells are
resistant to EGFR TKI (erlotinib) and have the weakest signals
amongst the lung cancer cell lines studied. These data indicate
that not only does EGFR:Grb2 PLA signal correspond to presence of
EGFR mutation status but it also may correlate with or predict
sensitivity to EGFR TKI.
[0057] FIG. 8 shows results of PLA examination of a tissue
microarray (TMA) that was produced from 21 FFPE lung cancer tumors,
14 without EGFR mutations and 7 with activating EGFR mutations.
Included in this array was normal spleen tissue as a negative
control. Tissues were examined using EGFR:Grb2 PLA. Of the 7 tumors
with EGFR mutation, 6 had the strongest signals across the sample
set. One tumor with EGFR mutation that lacked EGFR:Grb2 signal also
has a corresponding PIK3CA mutation leading to enhanced PIK3CA
activity. The 14 cases without EGFR mutation had lower degrees of
signal compared to the 6 positive cases with EGFR mutation.
Assuming a signal/noise cutoff of 10 (noise level =zero background
of spleen tissue), 6 of the 14 cases had positive levels of
EGFR:Grb2 PLA signal. This indicates a group of tumors lacking EGFR
mutation that nonetheless may have EGFR pathway activation.
[0058] FIGS. 9A and 9B show results of PLA assays revealing that
EGFR-GRB2 interactions are independent of expression and generally
correlated with EGFR phosphorylation. PLA assays were performed
across 9 non-small cell lung cancer (NSCLC) cell lines (PC9, H1650,
HCC827, H4006, H1299, H1648, H322, A549, and H23) and analyzed by
confocal microscopy at 400X magnification (FIG. 9A). Images
represent maximum projections of 12.times.0.76 .mu.m z-slices. Foci
are imaged in the far-red spectrum (Cy5) and nuclei were imaged in
the UV region with DAPI. Foci were detected in all EGFR mutant cell
lines, while several cell lines (H1299 and H23) have nearly
undetectable levels foci and serve as biological negative controls
demonstrating the specificity of PLA. Intermediate levels of foci
were detected in H322 and H1648, both of which exhibit some degree
of erlotinib sensitivity. These results show that EGFR-GRB2 foci
correlate with drug sensitivity. Immunoblot analysis of the 9 NSCLC
cell lines (50 .mu.g each) showed that all cells expressed
detectable levels of both EGFR and GRB2, while phosphorylation of
tyrosine 1068 of EGFR was detected only in EGFR mutant cell lines.
These results demonstrate that the presence of the EGFR and GRB2 is
necessary but not sufficient for interaction. The detection of
signal (visible foci) in drug sensitivity further supports the use
of PPI as a biomarker of drug sensitivity.
[0059] FIG. 10 shows PLA results revealing that EGFR-GRB2
interactions correlate with mutational status and are abrogated
with TKI treatment in vivo. Patient-derived xenografts were grown
in nude mice and treated with erlotinib for 28 days or with vehicle
control. Slides were prepared from excised tumor tissue (verified
by pathologist) and analyzed by PLA using 200.times. magnification.
Foci are imaged in the far-red spectrum (Cy5) and nuclei were
detected in the UV region with DAPI. Tumors derived from patients
with an activating EGFR mutation had very high levels of EGFR-GRB2
detectable by PLA, while EGFR wildtype tumors had markedly lower
levels. Importantly, PLA signal in the EGFR mutant tumors was
considerably reduced after administration of erlotinib. These
results indicate that mutation status is correlated with EGFR-GRB2
foci in vivo and that foci are correlated with erlotinib
activity.
[0060] FIG. 11 shows PLA revealing that normal tissues have low to
undetectable EGFR-GRB2 interactions. Eight normal tissues were
assayed by PLA for EGFR-GRB2 and imaged concordantly with staining
for the epithelial-origin marker cytokeratin using (Cy3). As
expected, low levels of foci are found in the trophoblasts in
placenta and epithelial cells of the gall bladder. Foci were not
detected in pancreas, spleen, colon, heart and normal lung. Thus,
EGFR-GRB2 interactions are detectable where EGFR is naturally
expressed, but at low levels consistent with the growth-arrested
state of most normal tissues.
[0061] FIGS. 12A-1-12D-2 show PLA results revealing EGFR-GRB2
interactions in lung tumor tissues and co-localization with
cytokeratin. Tissues were assayed by PLA in tumor microarray format
and analyzed as in FIG. 11. Foci are readily detectable in patient
2 (FIGS. 12B-1 and 12B-2) and patient 4 (FIGS. 12D-1 and 12D-2),
and absent in patient 1 (FIGS. 12A-1 and 12A-2) and patient 3
(FIGS. 12C-1 and 12C-2). Importantly, the foci observed in patients
2 and 4 are only found in cytokeratin-positive regions of the
specimen, indicating the specificity of PLA in tissues. For each
patient, the left panels (FIGS. 12A-1, 12B-1, 12C-1, and 12D-1)
show nuclei as blue (DAPI), cytokeratin as green (Cy3) and PLA foci
as red (Cy5). In each patient, the right panels (FIGS. 12A-2,
12B-2, 12C-2, and 12D-2) are the same images with the green channel
removed for clarity. These results demonstrate that PLA signals are
specific for epithelial-derived tissue and provide a tool to
quantify signal only within the tumor.
[0062] FIGS. 13A and 13B show EGFR-GRB2 PLA demonstrating that PPI
are independent of expression levels in lung tumor tissue. PLA were
performed and imaged as above on two patients, one harboring an
activating mutation in EGFR (FIG. 13A) and the other with wildtype
EGFR (FIG. 13B). Both patients express EGFR protein shown on the
left, where Cy5-labelled secondary antibodies were used to image
EGFR expression by immunofluorescence. Although patient B has
higher levels of EGFR protein expression (FIG. 13B), the PLA signal
is higher for patient A (FIG. 13A). These data suggest that PLA
signal may be a powerful indicator of EGFR signaling activity.
[0063] FIGS. 14A and 14B show PLA results demonstrating that PLA
foci can be visualized with immuno-fluorescence or brightfield
microscopy. PLA were performed on sequential tissue sections and
processed at the the same time. FIG. 14A was processed using
brightfield detection reagents (horseradish peroxidase-labeled
oligonucleotides) and counter-stained with hematoxylin. FIG. 14B
was processed using immunofluorescence detection reagents
(Cy5-labeled oligonucleotides) and nuclei stained with DAPI and
tissue autofluorescence imaged on the FITC channel. Images were
obtained at 400.times. magnification on brightfield or
immunofluorescent platforms, respectively. Similar results were
obtained with both methods of detection, indicating that PLA can be
performed using only light microscopy.
[0064] FIGS. 15A-15C show PLA results demonstrating that PLA can be
used to monitor phosphorylation status. PLA were performed in three
cell lines to assess phosphorylation dynamics as phosphorylation of
RTKs is required for binding of downstream effectors. A549 cells
were stimulated with 50 ng/ml EGF for various timepoints and
phosphorylation of EGFR was monitored by PLA using a single
antibody to phosphor-EGFR.sub.Y1068 (FIG. 15A). Unstimulated A549
cells have low levels of EGFR phosphorylation, which quickly
increases before diminishing. PC9 cells were treated for 3 hours
with erlotinib and assayed as above (FIG. 15B). 3 hours of
erlotinib treatment abolishes the phosphorylation of EGFR. For
FIGS. 15A and 15B, immunoblots confirmed the changes in
phosphorylation status (FIG. 15A-1 and 15B-1, respectively). H3122
cells were assayed by PLA using an antibody to ALK and total
phosphotyrosine to monitor the change in phosphorylation of ALK in
response to crizotinib (FIG. 15C). 3 hours of crizotinib treatment
abolishes the phosphorylation of ALK. Importantly, these assays
demonstrate that phosphorylation dynamics can be monitored by PLA
and that using an RTK antibody paired with a "pan" phosphotyrosine
is a viable approach.
[0065] FIGS. 16A-16D show results of ALK-Shcl PLA in EML4-ALK
rearranged lung cancer cells. FIGS. 16B and 16D show interaction
foci between ALK and Shcl in EML4-ALK rearranged lung cancer cells
that disappear with ALK TKI (crizotinib therapy). FIGS. 16A and 16C
show pTyr ALK.
[0066] FIG. 17 shows PLA results demonstrating that PLA facilitate
the observation of pharmacodynamic effects of kinase inhibitors on
signaling networks. Tumor xenografts were established in mice with
either H1650 (EGFR mutant) or H322 (EGFR wildtype), treated with or
without erlotinib and allowed to grow for 18 days. Formalin-fixed,
paraffin-embedded tumor tissue was analyzed by PLA for the
interaction of EGFR-GRB2 (left) and EGFR-SHC1 (right). Nuclei are
shown in blue, PLA signal is red and cytokeratin is green. For
clarity, identical images are shown with and without cytokeratin
staining for each tumor. H1650 cells exhibit high levels of
EGFR-GRB2 and considerably higher levels of EGFR-Shc1, consistent
with the presence of an activating EGFR mutation. H322 cells have
lower levels of EGFR interactions, but still show a reduction upon
TKI treatment. These data indicate that PLA signal is markedly
reduced upon TKI treatment, specifically within the tumor tissues
and can be used to evaluate target-specific drug efficacy in
vivo.
DETAILED DISCLOSURE OF THE INVENTION
[0067] Proximity ligation assay (PLA)-based biomarkers could be an
important future direction in personalized medicine. They could be
useful for cancer prognosis as well as decision making regarding
targeted therapy. The present invention includes assays for
protein-protein interactions (PPI) that can be used to determine,
for example, when an activated receptor tyrosine protein kinase
binds a signal transduction molecule such as GRB2. This assay uses
PLAs to quantitatively measure PPI in tissue and cell samples such
as that prepared for microscopy. The PLA typically involves primary
and secondary antibodies binding two separate proteins in a sample;
short nucleic acid strands are attached to the secondary antibodies
which are ligated into circles and then amplified and labeled,
e.g., with fluorescent probes. The inventors have established in
cell lines that EGFR-GRB2 PPIs distinguish populations of lung
cancer cells, are independent of EGFR expression level and
generally correlate with EGFR phosphorylation. The assay has also
been shown to work in FFPE patient tissue samples. The assay can be
used to determine if a patient's receptor tyrosine kinase pathway
is activated and, therefore, may be a good candidate for
intervention with a tyrosine kinase inhibitor.
[0068] The assays of the invention represent a platform technology
expandable to a larger spectrum of tyrosine kinases and possible
signal transduction docking proteins. The assays may be used as a
surrogate for tyrosine phosphorylation status, as tyrosine
phosphorylation is not easily discerned in FFPE slides without the
slides being prepared in a special manner. The assays of the
invention can read out pathway activation resulting from mutation,
gene amplification, or autocrine factors. Moreover, not all
tyrosine kinases have phosphorylation specific antibodies, and it
may be easier to make antibodies to the overall tyrosine kinase
protein and its adaptor proteins. Given the market of tyrosine
kinase inhibitors, both approved and in development, this
technology can provide a number of potential biomarker strategies
including diagnostics for drug response or companion
diagnostics.
[0069] One aspect of the invention concerns a method for assessing
the sensitivity of a malignancy to a treatment based on PPI. The
method for assessing the sensitivity of a malignancy to a treatment
comprises comparing a protein-protein interaction (PPI) profile
obtained from a sample of the malignancy to a reference PPI
profile. The sample PPI profile represents the abundance of target
binding partners that are in proximity to each other within the
sample. A negative result (lack of PPI) in a sample would indicate
a lack of sensitivity to the PPI modulator and be useful clinically
to avoid giving patients unnecessary PPI modulator therapy. A
positive result (presence of PPI) would indicate that the
malignancy is potentially sensitive to the PPI modulator and allow
the clinician to give the PPI modulator therapy to those patients
who would be most likely to benefit. As a specific example, the
method may be used for determining whether there is no EGF
signaling in a sample such as spleen tissue, and this kind of a
negative result can be useful clinically to avoid giving patients
unnecessary EGF receptor inhibitor therapy, or alternatively be
used to give the therapy to those patients who would be most likely
to benefit.
[0070] PPI profiles (e.g., sample PPI profile, and reference PPI
profile) may each be expressed as a value representative of the
abundance of target binding partners in proximity to each other
within the sample. The sample PPI profile and reference PPI profile
may be expressed by any method useful for comparison purposes, such
as a numeric value, score, cutoff (threshold), or other expression.
For example, a negative result in which a PPI profile in a sample
does not reach the cutoff would be useful clinically to avoid
giving patients unnecessary PPI modulator therapy. A positive
result in which a PPI profile in a sample is at or above the cutoff
would indicate potential sensitivity and allow the clinician to
give the PPI modulator therapy to those patients who would be most
likely to benefit.
[0071] In some embodiments of the methods and kits of the
invention, the target PPI is that of a known cancer signaling
network. Binding members of a target PPI may include protein
products of oncogenes or tumor suppressor genes, for example. In
some embodiments, the sample PPI profile represents the abundance
of target binding partners that are in proximity to each other
within the sample, wherein at least one of the target binding
partners is selected from among EGFR, ALK, MET, IGFR, Src, ErbB3,
Mig6, Grb2, Stsl, p85, and Hsp90.
[0072] In some embodiments of the methods and kits of the
invention, the target binding partners are selected from among EGFR
and ErbB3; EGFR and Grb2; EGFR and Mig6; EGFR and Sts1, EGFR and
Src; EGFR and Hsp90; ErbB3 and p85; ALK and EML4; MET and Gabl;
IGFR and IRS; Hsp90 and Cdc37; ALK and Shcl; and EGFR and Shc1.
[0073] In some embodiments of the methods and kits of the
invention, the PPI comprises a tyrosine kinase, such as the human
tyrosine kinases listed in Tables 3 and 4. In some embodiments, the
PPI comprises an interaction between the binding partners listed in
Table 5 (human tyrosine kinase interactions).
[0074] PLAs can be used to generate biomarkers against receptor
tyrosine kinases (RTKs). RTKs are important proteins in cancer and
highly "druggable" targets. Approximately twenty different RTK
classes have been identified thus far. In some embodiments of the
methods and kits of the invention, at least one of the target
binding partners is an RTK. In some embodiments of the methods and
kits of the invention, at least one of the target binding partners
is of an RTK class selected from among RTK class I (EGF receptor
family; ErbB family), RTK class II (insulin receptor family), RTK
class III (PDGF receptor family), RTK class IV (FGF receptor
family) RTK class V (VEGF receptors family), RTK class VI (HGF
receptor family), RTK class VII (Trk receptor family), RTK class
VIII (Eph receptor family), RTK class IX (AXL receptor family), RTK
class X (LTK receptor family), RTK class XI (TIE receptor family),
RTK class XII (ROR receptor family), XIII (DDR receptor family),
RTK class XIV (RET receptor family), RTK class XV (KLG receptor
family), RTK class XVI (RYK receptor family), and RTK class XVII
(MuSK receptor family).
[0075] In some embodiments of the methods and kits of the
invention, one or more of the binding partners of the target PPI
have one or more sequence mutations that are known to be associated
with occurrence of the malignancy. In other embodiments, the
binding partners of the target PPI do not harbor (lack) any
sequence mutations known to be associated with occurrence of the
malignancy, or with sensitivity to a treatment such as a PPI
modulator. In some embodiments, the binding partners of the target
PPI and the downstream effectors of the target PPI do not harbor
any sequence mutations known to be associated with occurrence of
the malignancy, or with sensitivity to a treatment such as a PPI
modulator. Such sequence aberrations in a subject or in a sample
can be detected using methods known in the art (e.g., mutation
analysis). Typically, abnormalities in nucleic acid sequences are
identified by comparison to reference sequence data (sequences of
normal cells or cancer cells) on databases, such as GenBank and
EMBL, and specific data resources such as Cancer Gene Census
(mutated genes causally implicated in human cancer), COSMIC
(Catalogue of Somatic Mutations in Cancer), and CGP Resequencing
Studies (somatic mutations from large scale resequencing of genes
in human cancer). Mutations causing or contributing to cancer may
be large-scale mutations, involving the deletion or addition of a
portion of a chromosome, or small-scale mutations, including point
mutations, deletions, insertions, which may occur in the promoter
region of a gene and affect its expression, may occur in the coding
sequence and alter the stability or function of the gene's protein
product.
[0076] The sample may be any cell sample potentially harboring the
target protein(s). For example, a cytology sample may be obtained
from a tissue selected from breast, ovaries, esophagus, stomach,
colon, rectum, anus, bile duct, brain, endometrium, lung, liver,
skin, prostate, kidney, nasopharynx, pancreas, head and neck,
kidney, lymphoma, leukemia, cervix, and bladder. The sample may be
a solid or non-solid tumor specimen. The tumor specimen may be a
carcinoma. The sample may be a new cancer, recurrent cancer,
primary cancer, or metastasized (secondary) cancer.
[0077] The sample may be obtained by methods known in the art, such
as surgery, biopsy, or from blood (e.g., circulating tumor cells),
ascites, or pleural effusion. The sample may be processed using
methods known in the art. For example, the sample may be fresh,
frozen, or formalin-fixed and paraffin-embedded (FFPE).
[0078] Preferably, the treatment against which the sample is being
assessed for sensistivity/resistance is a PPI modulator (i.e., a
PPI inhibitor or PPI inducer). However, the treatment may be a
treatment other than a PPI modulator.
[0079] Another aspect of the invention concerns a method for
treating a malignancy in a subject, comprising administering a
protein-protein interaction (PPI) modulator to the subject, wherein
the subject is predetermined to be sensitive to the PPI modulator
based on a PPI profile obtained from a sample of the malignancy. In
some embodiments, the PPI modulator is an inhibitor of the PPI of
the PPI profile (i.e., a PPI inhibitor). In some embodiments, the
PPI modulator is an inducer of the PPI of the PPI profile (i.e., a
PPI inducer).
[0080] Another aspect of the invention concerns a method for
treating a malignancy in a subject, comprising:
[0081] (a) assessing the sensitivity of a malignancy in the
subject, comprising comparing a protein-protein interaction (PPI)
profile obtained from a sample of the malignancy to a reference PPI
profile; and
[0082] (b) administering a PPI modulator to the subject if the
malignancy is assessed to be sensitive to the PPI modulator; and
withholding the PPI modulator from the subject if the malignancy is
assessed to be resistant to the PPI modulator. In some embodiments,
the PPI modulator is an inhibitor of the PPI of the PPI profile
(i.e., a PPI inhibitor). In some embodiments, the PPI modulator is
an inducer of the PPI of the PPI profile (i.e., a PPI inducer).
[0083] The invention includes also includes kits useful for
carrying out methods of the invention, (e.g., methods for the
classification of cancers as sensitive or resistant to treatments
based on protein-protein interactions, treatment of cancer,
identification of biomarkers, identification of protein-protein
interaction modulators, and selection of cancer treatments). Thus,
one aspect of the invention concerns a kit for detecting the
proximity of target binding partners within a cancer
protein-protein interaction network, comprising a primary antibody
to at least one of the target binding partners, and a proximity
probe comprising a secondary antibody (that binds to the primary
antibody) with an oligonucleotide conjugated thereto. In preferred
embodiments, the kit comprises:
[0084] a first primary antibody to a first target binding
partner;
[0085] a second primary antibody to a second target binding
partner;
[0086] a first proximity probe comprising a first secondary
antibody (that binds to the first primary antibody) with an
oligonucleotide conjugated thereto; and
[0087] a second proximity probe comprising a second secondary
antibody (that binds to the second primary antibody) with an
oligonucleotide conjugated thereto, wherein when the
oligonucleotides of the first and second proximity probes are in
sufficient proximity to each other, the oligonucleotides of the
proximity probes interact in the presence circle-forming
oligonucleotides by enzymatic ligation and form a circular product
that is amplified by rolling circle replication, producing an
amplification product. Optionally, the kit further comprises a
labeled oligonucleotide probe that hybridizes with the
amplification product, allowing detection and quantification of the
amplification product (representing the association (close
proximity) of the target binding partners).
[0088] Another aspect of the invention concerns a kit for detecting
the proximity of target binding partners within tertiary PPI (a
complex having three or more protein binding partners), referred to
herein as the "tertiary interaction kit". The target PPI may be
within a cancer signaling network, but is not limited to such
applications. The tertiary interaction kit can be used to study
viruses, for example, in which a viral protein forms an interaction
with protein A and protein B in a cell, giving rise to a tertiary
complex. Identification of this tertiary PPI may be diagnostic of
an active infection or yield important information about the
prognosis or predict the correct therapy. In cancer, a tertiary
complex may provide important information about the therapeutic
efficacy.
[0089] The tertiary interaction kit comprises:
[0090] a first primary antibody to a first target binding partner
of the tertiary interaction;
[0091] a second primary antibody to a second target binding partner
of the tertiary interaction;
[0092] a third primary antibody to a third target binding partner
of the tertiary interaction;
[0093] a first proximity probe comprising a first secondary
antibody (that binds to the first primary antibody) with an
oligonucleotide conjugated thereto; and
[0094] a second proximity probe comprising a second secondary
antibody (that binds to the second primary antibody) with an
oligonucleotide conjugated thereto;
[0095] a third proximity probe comprising a third secondary
antibody (that binds to the third primary antibody) with an
oligonucleotide conjugated thereto;
[0096] wherein when the oligonucleotides of the first and second
proximity probes are in sufficient proximity to each other, the
oligonucleotides of the first and second proximity probes interact
in the presence circle-forming oligonucleotides by enzymatic
ligation and form a circular product that is amplified by rolling
circle replication, producing a first amplification product;
and
[0097] wherein when the oligonucleotides of the second and third
proximity probes are in sufficient proximity to each other, the
oligonucleotides of the second and third proximity probes interact
in the presence of circle-forming oligonucleotides by enzymatic
ligation and form a circular product that is amplified by rolling
circle replication, producing a second amplification product.
Optionally, the kit detecting proximity of binding partners within
a tertiary PPI further comprises a first labeled oligonucleotide
probe that hybridizes with the first amplification product,
allowing detection and quantification of the first amplification
product (representing the association (close proximity) of the
first and second target binding partners), and a second labeled
oligonucleotide probe that hybridizes with the second amplification
product, allowing detection and quantification of the second
amplification product (representing the association of the second
and third target binding partners). Each primary antibody is of a
different species (e.g., mouse, rabbit, and goat, for example).
Preferably, the label of the first labeled oligonucleotide yields a
signal (e.g., a color) that is distinguishable from that of the
second labeled oligonucleotide (red and green, for example). An
additional primary antibody, proximity probe, and (optionally)
oligonucleotide probe can be included in the kit for each
additional target binding partner within the tertiary PPI to be
measured. Optionally, images can be constructed for visualization
showing each signal with overlay (e.g., red and green overlay).
[0098] In the various kits of the invention, each kit can include
instructions or packaging materials that describe how to use a
compound or composition (e.g., a reagent such as a primary
antibody, a secondary antibody, a labeled oligonucleotide probe
that hybridizes with the amplification product) of the kit. Within
the kit, the secondary antibody may be uncojugated or conjugated to
an oligonucleotide (making the secondary antibody a proximity
probe). The kits may also comprise, e.g., polymerase (for the
amplification reaction), ligase (for the ligation reaction), a
buffering agent, a preservative, or a protein stabilizing agent.
The kits may also comprise components necessary for detecting the
label (e.g., an enzyme or substrate). The kit may also contain a
control sample or a series of control samples that can be assayed
and compared to a test sample. Each kit can include one or more
containers for individually enclosing each component of the kit.
Containers of the kits can be of any suitable material, e.g.,
glass, plastic, metal, etc., and of any suitable size, shape, or
configuration. The one or more containers are can be enclosed
within outer packaging.
[0099] The kits of the invention may be used by contacting the
cell-containing sample with the primary antibodies (allowing the
primary antibodies to bind to their respective protein targets),
contacting the sample with the proximity probes (allowing the
proximity probes to bind to their respective antibody targets),
allowing the nucleic acid strands (also referred to as arms) of the
proximity probes to ligate if binding in close proximity (adding
ligase if necessary), amplifying the ligation product (adding
polymerase if necessary) to produce an amplification product, and
measuring the amplification product corresponding to the
interaction of each pair of protein targets within the PPI.
Measurement of the amplification product can be made using methods
known in the art for detecting and quantifying nucleic acid
amplification products, e.g., by adding a labeled oligonucleotide
that hybridizes to a sequence of the amplification product, and
analyzing the sample by visualizing the amplification product (as
represented by the label signal) with an appropriate visualization
device, such as a confocal or fluorescence microscope.
[0100] Another aspect of the invention concerns a method for
measuring protein-protein interactions having three or more binding
partners (a tertiary interaction) in a sample, comprising:
[0101] contacting the sample with three or more primary antibodies
to three or more corresponding target binding partners within a
target tertiary interaction;
[0102] contacting the sample with three or more proximity probes
(first, second, and third proximity probes) comprising a secondary
antibody that binds to the corresponding first antibody, wherein
each proximity probe has an oligonucleotide conjugated thereto;
[0103] wherein when the oligonucleotides of the first and second
proximity probes are in sufficient proximity to each other, the
oligonucleotides of the first and second proximity probes interact
in the presence circle-forming oligonucleotides by enzymatic
ligation and form a circular product that is amplified by rolling
circle replication, producing a first amplification product; and
[0104] wherein when the oligonucleotides of the second and third
proximity probes are in sufficient proximity to each other, the
oligonucleotides of the second and third proximity probes interact
in the presence of circle-forming oligonucleotides by enzymatic
ligation and form a circular product that is amplified by rolling
circle replication, producing a second amplification product; and
measuring the first and second amplification products.
[0105] In some embodiments, measurement of the first and second
amplification products comprises contacting the sample with two or
more labeled oligonucleotides, comprising a first labeled
oligonucleotide that hybridizes to a sequence of the first
amplification product, and a second labeled oligonucleotide that
hybridizes to a sequence of the second amplification product, to
produce labeled amplification products, wherein the labels are
distinguishable from one another; and measuring the PPIs by
visualizing the labeled amplification products. An additional
primary antibody, proximity probe, and labeled oligonucleotide can
be used for each additional target binding partner within the
tertiary PPI.
[0106] Another aspect of the invention concerns a method for the
identification of a biomarker, comprising selecting two or more
target binding partners within a cancer sample; generating a PPI
profile for the two or more target binding partners; and comparing
the PPI profile to the responsiveness of the cancer to a treatment
in vitro and/or in vivo (for example, in xenograft animal models or
human subjects). Correlation between the PPI profile to the
responsiveness of the cancer to a treatment of the cancer in vitro
and/or in vivo is indicative of a biomarker for treatment
responsiveness for the cancer. In some embodiments, the treatment
is a kinase inhibitor (e.g., a tyrosine kinase inhibitor (TKI)). In
some embodiments, at least one of the two or more target binding
partners comprises a receptor tyrosine kinase (RTK). In some
embodiments, RTK is within RTK class I-XVII. Preferably,
responsiveness of the treatment to the cancer in vitro, or in vivo
with xenograft animal models, is determined with the subject's
cancer cells; however, cancer cell lines known to be predictive of
responsiveness to the subject's cancer type may be utilized to
compare with the PPI profile.
[0107] Another aspect of the invention concerns a method for
identifying an agent as a PPI modulator, comprising: contacting
cancer cells with a candidate agent in vitro or in vivo; and
determining whether the candidate agent modulates a selected PPI in
a sample of the cancer cells. The candidate agent may be any
substance that potentially modulates (increases, decreases, or
otherwise alters) the PPI. The candidate agent may be a small
molecule, polypeptide, or nucleic acid, for example. Determination
of changes in PPI can be made by comparing a first PPI profile of
the cancer cells obtained prior to the contacting step to a second
PPI profile of the cancer cells obtained after the contacting step,
wherein a change in the PPI is indicative of a PPI modulator.
[0108] By inhibiting the growth of cells proliferating in an
aberrant manner, the methods, PPI modulators, and compositions of
the present invention can be used to treat a number of cell
proliferation disorders, such as cancers, including, but not
limited to, leukemias and lymphomas, such as acute lymphocytic
leukemia, acute non-lymphocytic leukemias, chronic lymphocytic
leukemia, chronic myelogenous leukemia, Hodgkin's Disease,
non-Hodgkin's lymphomas, and multiple myeloma, childhood solid
tumors such as brain tumors, neuroblastoma, retinoblastoma, Wilms'
Tumor, bone tumors, and soft-tissue sarcomas, common solid tumors
of adults such as lung cancer, colon and rectum cancer, breast
cancer, prostate cancer, urinary cancers, uterine cancers, bladder
cancers, oral cancers, pancreatic cancer, melanoma and other skin
cancers, stomach cancer, ovarian cancer, brain tumors, liver
cancer, laryngeal cancer, thyroid cancer, esophageal cancer, and
testicular cancer. The methods of the subject invention can be
carried out in vivo or in vitro, to inhibit the growth of cells
(e.g., cancer cells) in humans and non-human mammals.
[0109] In some embodiments, the proliferation disorder to be
treated is a cancer producing a tumor characterized by aberrant
protein-protein interaction.
[0110] The methods of the present invention can be advantageously
combined with at least one additional treatment method, including
but not limited to, surgery, chemotherapy, radiation therapy, or
any other therapy known to those of skill in the art for the
treatment and management of proliferation disorders such as
cancer.
[0111] While PPI modulators can be administered to cells in vitro
and in vivo as isolated agents, it is preferred to administer PPI
modulators as part of a pharmaceutical composition, in association
with at least one pharmaceutically acceptable carrier. The
pharmaceutical composition can be adapted for various routes of
administration, such as enteral, parenteral, intravenous,
intramuscular, topical, subcutaneous, and so forth. Administration
can be continuous or at distinct intervals, as can be determined by
a person of ordinary skill in the art.
[0112] The PPI modulators utilized in the invention can be
formulated according to known methods for preparing
pharmaceutically useful compositions. Formulations are described in
a number of sources which are well known and readily available to
those skilled in the art. For example, Remington's Pharmaceutical
Science (Martin, E. W., 1995, Easton Pa., Mack Publishing Company,
19.sup.th ed.) describes formulations which can be used in
connection with the subject invention. Formulations suitable for
administration include, for example, aqueous sterile injection
solutions, which may contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient; and aqueous and nonaqueous sterile
suspensions which may include suspending agents and thickening
agents. The formulations may be presented in unit-dose or
multi-dose containers, for example sealed ampoules and vials, and
may be stored in a freeze dried (lyophilized) condition requiring
only the condition of the sterile liquid carrier, for example,
water for injections, prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powder,
granules, tablets, etc. It should be understood that in addition to
the ingredients particularly mentioned above, the compositions of
the subject invention can include other agents conventional in the
art having regard to the type of formulation in question.
[0113] Examples of pharmaceutically acceptable salts are organic
acid addition salts formed with acids that form a physiological
acceptable anion, for example, tosylate, methanesulfonate, acetate,
citrate, malonate, tartarate, succinate, benzoate, ascorbate,
alpha-ketoglutarate, and alpha-glycerophosphate. Suitable inorganic
salts may also be formed, including hydrochloride, sulfate,
nitrate, bicarbonate, and carbonate salts.
[0114] Pharmaceutically acceptable salts of compounds may be
obtained using standard procedures well known in the art, for
example, by reacting a sufficiently basic compound such as an amine
with a suitable acid affording a physiologically acceptable anion.
Alkali metal (for example, sodium, potassium or lithium) or
alkaline earth metal (for example calcium) salts of carboxylic
acids can also be made.
[0115] As used herein, the term "analogs" refers to compounds which
are substantially the same as another compound but which may have
been modified by, for example, adding side groups, oxidation or
reduction of the parent structure. Analogs of particular PPIs, and
other agents disclosed herein, can be readily prepared using
commonly known standard reactions. These standard reactions
include, but are not limited to, hydrogenation, alkylation,
acetylation, and acidification reactions. Chemical modifications
can be accomplished by those skilled in the art by protecting all
functional groups present in the molecule and deprotecting them
after carrying out the desired reactions using standard procedures
known in the scientific literature (Greene, T. W. and Wuts, P. G.
M. "Protective Groups in Organic Synthesis" John Wiley & Sons,
Inc. New York. 3rd Ed. pg. 819, 1999; Honda, T. et al. Bioorg. Med.
Chem. Lett., 1997, 7:1623-1628; Honda, T. et al. Bioorg. Med. Chem.
Lett., 1998, 8:2711-2714; Konoike, T. et al. J. Org. Chem., 1997,
62:960-966; Honda, T. et al. J. Med. Chem., 2000, 43:4233-4246;
each of which are hereby incorporated herein by reference in their
entirety). Analogs, fragments, and variants of PPI modulators
exhibiting the desired biological activity (such as induction of
cell death, cytotoxicity, cytostaticity, induction of cell cycle
arrest, etc.) can be identified or confirmed using cellular assays
or other in vitro or in vivo assays.
[0116] Therapeutic application of the PPI modulators and
compositions comprising them can be accomplished by any suitable
therapeutic method and technique presently or prospectively known
to those skilled in the art.
[0117] Active agents such as PPI modulators may be locally
administered at one or more anatomical sites, such as sites of
unwanted cell growth (such as a tumor site, e.g., injected or
topically applied to the tumor), optionally in combination with a
pharmaceutically acceptable carrier such as an inert diluent. PPI
modulators may be systemically administered, such as intravenously
or orally, optionally in combination with a pharmaceutically
acceptable carrier such as an inert diluent, or an assimilable
edible carrier for oral delivery. They may be enclosed in hard or
soft shell gelatin capsules, may be compressed into tablets, or may
be incorporated directly with the food of the patient's diet. For
oral therapeutic administration, the PPI modulators may be combined
with one or more excipients and used in the form of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, aerosol sprays, and the like.
[0118] The tablets, troches, pills, capsules, and the like may also
contain the following: binders such as gum tragacanth, acacia, corn
starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, fructose, lactose or aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring may be added. When the unit dosage form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a vegetable oil or a polyethylene glycol. Various
other materials may be present as coatings or to otherwise modify
the physical form of the solid unit dosage form. For instance,
tablets, pills, or capsules may be coated with gelatin, wax,
shellac, or sugar and the like. A syrup or elixir may contain the
active compound, sucrose or fructose as a sweetening agent, methyl
and propylparabens as preservatives, a dye and flavoring such as
cherry or orange flavor. Of course, any material used in preparing
any unit dosage form should be pharmaceutically acceptable and
substantially non-toxic in the amounts employed. In addition, the
PPI modulators may be incorporated into sustained-release
preparations and devices.
[0119] The active agent (e.g., a PPI modulator) may also be
administered intravenously or intraperitoneally by infusion or
injection. Solutions of the active agent can be prepared in water,
optionally mixed with a nontoxic surfactant. Dispersions can also
be prepared in glycerol, liquid polyethylene glycols, triacetin,
and mixtures thereof and in oils. Under ordinary conditions of
storage and use, these preparations can contain a preservative to
prevent the growth of microorganisms.
[0120] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the active agent (e.g., PPI modulator)
which are adapted for the extemporaneous preparation of sterile
injectable or infusible solutions or dispersions, optionally
encapsulated in liposomes. The ultimate dosage form should be
sterile, fluid and stable under the conditions of manufacture and
storage. The liquid carrier or vehicle can be a solvent or liquid
dispersion medium comprising, for example, water, ethanol, a polyol
(for example, glycerol, propylene glycol, liquid polyethylene
glycols, and the like), vegetable oils, nontoxic glyceryl esters,
and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the formation of liposomes, by the
maintenance of the required particle size in the case of
dispersions or by the use of surfactants. Optionally, the
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars, buffers or sodium chloride. Prolonged absorption
of the injectable compositions can be brought about by the
inclusion of agents that delay absorption, for example, aluminum
monostearate and gelatin.
[0121] Sterile injectable solutions are prepared by incorporating
the PPI modulators above, as required, followed by filter
sterilization. In the case of sterile powders for the preparation
of sterile injectable solutions, the preferred methods of
preparation are vacuum drying and the freeze drying techniques,
which yield a powder of the active ingredient plus any additional
desired ingredient present in the previously sterile-filtered
solutions.
[0122] For topical administration, active agents such as PPI
modulators may be applied in pure-form, i.e., when they are
liquids. However, it will generally be desirable to administer them
topically to the skin as compositions, in combination with a
dermatologically acceptable carrier, which may be a solid or a
liquid.
[0123] The PPI modulators can be applied topically to a subject's
skin to reduce the size (and may include complete removal) of
malignant or benign growths. The PPI modulators can be applied
directly to the growth. Preferably, the PPI is applied to the
growth in a formulation such as an ointment, cream, lotion,
solution, tincture, or the like. Drug delivery systems for delivery
of pharmacological substances to dermal lesions can also be used,
such as that described in U.S. Pat. No. 5,167,649 (Zook).
[0124] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, alcohols or glycols or
water-alcohol/glycol blends, in which the active agent can be
dissolved or dispersed at effective levels, optionally with the aid
of non-toxic surfactants. Adjuvants such as fragrances and
additional antimicrobial agents can be added to optimize the
properties for a given use. The resultant liquid compositions can
be applied from absorbent pads, used to impregnate bandages and
other dressings, or sprayed onto the affected area using pump-type
or aerosol sprayers, for example.
[0125] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user. Examples of
useful dermatological compositions which can be used to deliver the
active agent to the skin are disclosed in Jacquet et al. (U.S. Pat.
No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S.
Pat. No. 4,559,157) and Woltzman (U.S. Pat. No. 4,820,508).
[0126] Useful dosages of the pharmaceutical compositions of the
present invention can be determined by comparing their in vitro
activity, and in vivo activity in animal models. Methods for the
extrapolation of effective dosages in mice, and other animals, to
humans are known to the art; for example, see U.S. Pat. No.
4,938,949.
[0127] Accordingly, pharmaceutical compositions can comprise PPI
modulator in combination with a pharmaceutically acceptable
carrier. Pharmaceutical compositions adapted for oral, topical or
parenteral administration, comprising an amount of an PPI
modulator, constitute a preferred embodiment of the invention. The
dose administered to a patient, particularly a human, in the
context of the present invention should be sufficient to achieve a
therapeutic response in the patient over a reasonable time frame,
without lethal toxicity, and preferably causing no more than an
acceptable level of side effects or morbidity. One skilled in the
art will recognize that dosage will depend upon a variety of
factors including the condition (health) of the subject, the body
weight of the subject, kind of concurrent treatment, if any,
frequency of treatment, therapeutic ratio, as well as the severity
and stage of the pathological condition.
[0128] Depending upon the disorder or disease condition to be
treated, a suitable dose(s) may be that amount that will reduce
proliferation or growth of the target cell(s), or induce cell
death. In the context of cancer, a suitable dose(s) is that which
will result in a concentration of the active agent (e.g., one or
more PPI modulators) in cancer tissue, such as a malignant tumor,
which is known to achieve the desired response. The preferred
dosage is the amount which results in maximum inhibition of cancer
cell growth, without unmanageable side effects. Administration
active agents, such as PPI modulators, can be continuous or at
distinct intervals, as can be determined by a person of ordinary
skill in the art.
[0129] To provide for the administration of such dosages for the
desired therapeutic treatment, in some embodiments, pharmaceutical
compositions of the invention can comprise between about 0.1% and
45%, and especially, 1 and 15%, by weight of the total of one or
more of the compounds of the invention based on the weight of the
total composition including carrier or diluents. Illustratively,
dosage levels of the administered active ingredients can be:
intravenous, 0.01 to about 20 mg/kg; intraperitoneal, 0.01 to about
100 mg/kg; subcutaneous, 0.01 to about 100 mg/kg; intramuscular,
0.01 to about 100 mg/kg; orally 0.01 to about 200 mg/kg, and
preferably about 1 to 100 mg/kg; intranasal instillation, 0.01 to
about 20 mg/kg; and aerosol, 0.01 to about 20 mg/kg of animal
(body) weight.
[0130] Mammalian species which benefit from the disclosed methods
include, but are not limited to, primates, such as apes,
chimpanzees, orangutans, humans, monkeys; domesticated animals
(e.g., pets) such as dogs, cats, guinea pigs, hamsters, Vietnamese
pot-bellied pigs, rabbits, and ferrets; domesticated farm animals
such as cows, buffalo, bison, horses, donkey, swine, sheep, and
goats; exotic animals typically found in zoos, such as bear, lions,
tigers, panthers, elephants, hippopotamus, rhinoceros, giraffes,
antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,
koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals,
sea lions, elephant seals, otters, porpoises, dolphins, and whales.
Other species that may benefit from the disclosed methods include
fish, amphibians, avians, and reptiles. As used herein, the terms
"patient", "subject", and "individual" are used interchangeably and
are intended to include such human and non-human species. Likewise,
in vitro methods of the present invention can be carried out on
cells of such human and non-human species. In some embodiments, the
cells are obtained from a subject. In other embodiments, the cells
are cells of a cancer cell line.
[0131] Patients in need of treatment using the methods of the
present invention can be identified using standard techniques known
to those in the medical or veterinary professions, as
appropriate.
[0132] The terms "cancer" and "malignancy" are used herein
interchangeably to refer to or describe the physiological condition
in mammals that is typically characterized by unregulated cell
growth. The cancer may be multi-drug resistant (MDR) or
drug-sensitive. Examples of cancer include but are not limited to,
carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More
particular examples of such cancers include breast cancer, prostate
cancer, colon cancer, squamous cell cancer, small-cell lung cancer,
non-small cell lung cancer, gastrointestinal cancer, pancreatic
cancer, cervical cancer, ovarian cancer, peritoneal cancer, liver
cancer, e.g., hepatic carcinoma, bladder cancer, colorectal cancer,
endometrial carcinoma, kidney cancer, and thyroid cancer.
[0133] Other non-limiting examples of cancers are basal cell
carcinoma, biliary tract cancer; bone cancer; brain and CNS cancer;
choriocarcinoma; connective tissue cancer; esophageal cancer; eye
cancer; cancer of the head and neck; gastric cancer;
intra-epithelial neoplasm; larynx cancer; lymphoma including
Hodgkin's and Non-Hodgkin's lymphoma; melanoma; myeloma;
neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and
pharynx); retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer
of the respiratory system; sarcoma; skin cancer; stomach cancer;
testicular cancer; uterine cancer; cancer of the urinary system, as
well as other carcinomas and sarcomas. Examples of cancer types
that may potentially be sampled and treated using the kits and
methods of the invention are also listed in Table 1.
TABLE-US-00001 TABLE 1 Examples of Cancer Types Acute Lymphoblastic
Leukemia, Adult Hairy Cell Leukemia Acute Lymphoblastic Leukemia,
Head and Neck Cancer Childhood Hepatocellular (Liver) Cancer, Adult
Acute Myeloid Leukemia, Adult (Primary) Acute Myeloid Leukemia,
Childhood Hepatocellular (Liver) Cancer, Childhood Adrenocortical
Carcinoma (Primary) Adrenocortical Carcinoma, Childhood Hodgkin's
Lymphoma, Adult AIDS-Related Cancers Hodgkin's Lymphoma, Childhood
AIDS-Related Lymphoma Hodgkin's Lymphoma During Pregnancy Anal
Cancer Hypopharyngeal Cancer Astrocytoma, Childhood Cerebellar
Hypothalamic and Visual Pathway Glioma, Astrocytoma, Childhood
Cerebral Childhood Basal Cell Carcinoma Intraocular Melanoma Bile
Duct Cancer, Extrahepatic Islet Cell Carcinoma (Endocrine Pancreas)
Bladder Cancer Kaposi's Sarcoma Bladder Cancer, Childhood Kidney
(Renal Cell) Cancer Bone Cancer, Osteosarcoma/Malignant Kidney
Cancer, Childhood Fibrous Histiocytoma Laryngeal Cancer Brain Stem
Glioma, Childhood Laryngeal Cancer, Childhood Brain Tumor, Adult
Leukemia, Acute Lymphoblastic, Adult Brain Tumor, Brain Stem
Glioma, Leukemia, Acute Lymphoblastic, Childhood Childhood
Leukemia, Acute Myeloid, Adult Brain Tumor, Cerebellar Astrocytoma,
Leukemia, Acute Myeloid, Childhood Childhood Leukemia, Chronic
Lymphocytic Brain Tumor, Cerebral Leukemia, Chronic Myelogenous
Astrocytoma/Malignant Glioma, Leukemia, Hairy Cell Childhood Lip
and Oral Cavity Cancer Brain Tumor, Ependymoma, Childhood Liver
Cancer, Adult (Primary) Brain Tumor, Medulloblastoma, Liver Cancer,
Childhood (Primary) Childhood Lung Cancer, Non-Small Cell Brain
Tumor, Supratentorial Primitive Lung Cancer, Small Cell
Neuroectodermal Tumors, Childhood Lymphoma, AIDS-Related Brain
Tumor, Visual Pathway and Lymphoma, Burkitt's Hypothalamic Glioma,
Childhood Lymphoma, Cutaneous T-Cell, see Mycosis Brain Tumor,
Childhood Fungoides and Sezary Syndrome Breast Cancer Lymphoma,
Hodgkin's, Adult Breast Cancer, Childhood Lymphoma, Hodgkin's,
Childhood Breast Cancer, Male Lymphoma, Hodgkin's During Pregnancy
Bronchial Adenomas/Carcinoids, Lymphoma, Non-Hodgkin's, Adult
Childhood Lymphoma, Non-Hodgkin's, Childhood Burkitt's Lymphoma
Lymphoma, Non-Hodgkin's During Carcinoid Tumor, Childhood Pregnancy
Carcinoid Tumor, Gastrointestinal Lymphoma, Primary Central Nervous
System Carcinoma of Unknown Primary Macroglobulinemia,
Waldenstrom's Central Nervous System Lymphoma, Malignant Fibrous
Histiocytoma of Primary Bone/Osteosarcoma Cerebellar Astrocytoma,
Childhood Medulloblastoma, Childhood Cerebral Astrocytoma/Malignant
Melanoma Glioma, Childhood Melanoma, Intraocular (Eye) Cervical
Cancer Merkel Cell Carcinoma Childhood Cancers Mesothelioma, Adult
Malignant Chronic Lymphocytic Leukemia Mesothelioma, Childhood
Chronic Myelogenous Leukemia Metastatic Squamous Neck Cancer with
Chronic Myeloproliferative Disorders Occult Primary Colon Cancer
Multiple Endocrine Neoplasia Syndrome, Colorectal Cancer, Childhood
Childhood Cutaneous T-Cell Lymphoma, see Multiple Myeloma/Plasma
Cell Neoplasm Mycosis Fungoides and Sezary Mycosis Fungoides
Syndrome Myelodysplastic Syndromes Endometrial Cancer
Myelodysplastic/Myeloproliferative Diseases Ependymoma, Childhood
Myelogenous Leukemia, Chronic Esophageal Cancer Myeloid Leukemia,
Adult Acute Esophageal Cancer, Childhood Myeloid Leukemia,
Childhood Acute Ewing's Family of Tumors Myeloma, Multiple
Extracranial Germ Cell Tumor, Myeloproliferative Disorders, Chronic
Childhood Nasal Cavity and Paranasal Sinus Cancer Extragonadal Germ
Cell Tumor Nasopharyngeal Cancer Extrahepatic Bile Duct Cancer
Nasopharyngeal Cancer, Childhood Eye Cancer, Intraocular Melanoma
Neuroblastoma Eye Cancer, Retinoblastoma Non-Hodgkin's Lymphoma,
Adult Gallbladder Cancer Non-Hodgkin's Lymphoma, Childhood Gastric
(Stomach) Cancer Non-Hodgkin's Lymphoma During Pregnancy Gastric
(Stomach) Cancer, Childhood Non-Small Cell Lung Cancer
Gastrointestinal Carcinoid Tumor Oral Cancer, Childhood Germ Cell
Tumor, Extracranial, Oral Cavity Cancer, Lip and Childhood
Oropharyngeal Cancer Germ Cell Tumor, Extragonadal
Osteosarcoma/Malignant Fibrous Germ Cell Tumor, Ovarian
Histiocytoma of Bone Gestational Trophoblastic Tumor Ovarian
Cancer, Childhood Glioma, Adult Ovarian Epithelial Cancer Glioma,
Childhood Brain Stem Ovarian Germ Cell Tumor Glioma, Childhood
Cerebral Ovarian Low Malignant Potential Tumor Astrocytoma
Pancreatic Cancer Glioma, Childhood Visual Pathway and Pancreatic
Cancer, Childhood Hypothalamic Pancreatic Cancer, Islet Cell Skin
Cancer (Melanoma) Paranasal Sinus and Nasal Cavity Cancer Skin
Carcinoma, Merkel Cell Parathyroid Cancer Small Cell Lung Cancer
Penile Cancer Small Intestine Cancer Pheochromocytoma Soft Tissue
Sarcoma, Adult Pineoblastoma and Supratentorial Primitive Soft
Tissue Sarcoma, Childhood Neuroectodermal Tumors, Childhood
Squamous Cell Carcinoma, see Skin Pituitary Tumor Cancer
(non-Melanoma) Plasma Cell Neoplasm/Multiple Myeloma Squamous Neck
Cancer with Occult Pleuropulmonary Blastoma Primary, Metastatic
Pregnancy and Breast Cancer Stomach (Gastric) Cancer Pregnancy and
Hodgkin's Lymphoma Stomach (Gastric) Cancer, Childhood Pregnancy
and Non-Hodgkin's Lymphoma Supratentorial Primitive Primary Central
Nervous System Lymphoma Neuroectodermal Tumors, Childhood Prostate
Cancer T-Cell Lymphoma, Cutaneous, see Rectal Cancer Mycosis
Fungoides and Sezary Renal Cell (Kidney) Cancer Syndrome Renal Cell
(Kidney) Cancer, Childhood Testicular Cancer Renal Pelvis and
Ureter, Transitional Cell Thymoma, Childhood Cancer Thymoma and
Thymic Carcinoma Retinoblastoma Thyroid Cancer Rhabdomyosarcoma,
Childhood Thyroid Cancer, Childhood Salivary Gland Cancer
Transitional Cell Cancer of the Renal Salivary Gland Cancer,
Childhood Pelvis and Ureter Sarcoma, Ewing's Family of Tumors
Trophoblastic Tumor, Gestational Sarcoma, Kaposi's Unknown Primary
Site, Carcinoma of, Sarcoma, Soft Tissue, Adult Adult Sarcoma, Soft
Tissue, Childhood Unknown Primary Site, Cancer of, Sarcoma, Uterine
Childhood Sezary Syndrome Unusual Cancers of Childhood Skin Cancer
(non-Melanoma) Ureter and Renal Pelvis, Transitional Skin Cancer,
Childhood Cell Cancer Urethral Cancer Uterine Cancer, Endometrial
Uterine Sarcoma Vaginal Cancer Visual Pathway and Hypothalamic
Glioma, Childhood Vulvar Cancer Waldenstrom's Macroglobulinemia
Wilms' Tumor
[0134] As used herein, the term "tumor" refers to all neoplastic
cell growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. For example, a
particular cancer may be characterized by a solid mass tumor or
non-solid tumor. The solid tumor mass, if present, may be a primary
tumor mass. A primary tumor mass refers to a growth of cancer cells
in a tissue resulting from the transformation of a normal cell of
that tissue. In most cases, the primary tumor mass is identified by
the presence of a cyst, which can be found through visual or
palpation methods, or by irregularity in shape, texture or weight
of the tissue. However, some primary tumors are not palpable and
can be detected only through medical imaging techniques such as
X-rays (e.g., mammography) or magnetic resonance imaging (MM), or
by needle aspirations. The use of these latter techniques is more
common in early detection. Molecular and phenotypic analysis of
cancer cells within a tissue can usually be used to confirm if the
cancer is endogenous to the tissue or if the lesion is due to
metastasis from another site. The treatment methods of the
invention can be utilized for early, middle, or late stage disease,
and acute or chronic disease.
[0135] According to methods of the subject invention, a PPI
modulator can be administered to a patient by itself, or
co-administered with one or more other agents such as another PPI
modulator, or a different agent or agents. Co-administration can be
carried out simultaneously (in the same or separate formulations)
or consecutively. Furthermore, according to the method of the
subject invention, PPI modulators can be administered to a patient
as adjuvant therapy. For example, PPI modulators can be
administered to a patient in conjunction with chemotherapy,
radiation therapy, surgery, or a combination of two or more of the
foregoing.
[0136] Thus, the PPI modulators, whether administered separately,
or as a pharmaceutical composition, can include various other
components as additives. Examples of acceptable components or
adjuncts which can be employed in relevant circumstances include
antioxidants, free radical scavenging agents, peptides, growth
factors, antibiotics, bacteriostatic agents, immunosuppressives,
anticoagulants, buffering agents, anti-inflammatory agents,
anti-angiogenics, anti-pyretics, time-release binders, anesthetics,
steroids, and corticosteroids. Such components can provide
additional therapeutic benefit, act to affect the therapeutic
action of the compounds of the invention, or act towards preventing
any potential side effects which may be posed as a result of
administration of the compounds. The PPI modulators can be
conjugated to a therapeutic agent, as well.
[0137] Additional agents that can be co-administered to target
cells in vitro or in vivo, such as in a patient, in the same or as
a separate formulation, include those that modify a given
biological response, such as immunomodulators. For example,
proteins such as tumor necrosis factor (TNF), interferon (such as
alpha-interferon and beta-interferon), nerve growth factor (NGF),
platelet derived growth factor (PDGF), and tissue plasminogen
activator can be administered. Biological response modifiers, such
as lymphokines, interleukins (such as interleukin-1 (IL-1),
interleukin-2 (IL-2), and interleukin-6 (IL-6)), granulocyte
macrophage colony stimulating factor (GM-C SF), granulocyte colony
stimulating factor (G-CSF), or other growth factors can be
administered. In one embodiment, the methods and compositions of
the invention incorporate one or more agents selected from the
group consisting of anti-cancer agents, cytotoxic agents,
chemotherapeutic agents, anti-signaling agents, and anti-angiogenic
agents.
[0138] In some embodiments of the methods of the invention, at
least one additional anti-cancer agent (e.g., a chemotherapeutic
agent) is administered with the PPI modulator. In some embodiments,
the anti-cancer agent is selected from among suberoylanilide
hydroxamic acid (SAHA) or other histone deacetylase inhibitor,
arsenic trioxide, doxorubicin or other anthracycline DNA
intercalating agent, and etoposide or other topoisomerase II
inhibitor.
[0139] Within certain aspects of the present invention, one or more
PPI modulators as described herein may be present within a
pharmaceutical composition. A pharmaceutical composition comprises
one or more PPI modulators in combination with one or more
pharmaceutically or physiologically acceptable carriers, diluents
or excipients. Such compositions may comprise buffers (e.g.,
neutral buffered saline or phosphate buffered saline),
carbohydrates (e.g., glucose, mannose, sucrose or dextrans),
mannitol, proteins, polypeptides or amino acids such as glycine,
antioxidants, chelating agents such as EDTA or glutathione,
adjuvants (e.g., aluminum hydroxide) and/or preservatives. Within
yet other embodiments, compositions of the present invention may be
formulated as a lyophilizate. A PPI modulator may, but need not, be
encapsulated within liposomes using well known technology.
Compositions of the present invention may be formulated for any
appropriate manner of administration, including for example,
topical, oral, nasal, intravenous, intracranial, intraperitoneal,
subcutaneous, or intramuscular administration. For certain topical
applications, formulation as a cream or lotion, using well known
components, is preferred.
[0140] Various techniques may be utilized to facilitate delivery of
the PPI modulators to the target cells in vitro (including ex vivo)
and in vivo (Cellular Drug Delivery: Principles and Practice,
edited by Lu, D. R. and Oie, S., Human Press, Totowa, N.J., 2004).
Optionally, it may be desirable to facilitate delivery of the PPI
modulators through the outer cell membrane. Various carrier
molecules may be coupled to the PPI modulators to assist
penetration through biological membranes. For example, small
regions (e.g., 9-16 amino acids) of proteins called protein
transduction domains (PTDs) cell penetrating peptides (CPP) possess
the ability to traverse biological membranes through protein
transduction (Barnett, E. M. et al., Invest. Opthalmol. Vis. Sci.,
2006, 47:2589-2595; Schwarze S. R. et al., Science, 1999,
285(5433):1569-1572; Wadia, J. S. and Dowdy, S. F., Advanced Drug
Delivery Reviews, 2005, 57(4): 579-596; Wadia, J. S. and Dowdy, S.
F., Curr. Opin. Biotechnol., 2002, 13(1)52-56; Ho A. et al., Cancer
Research, 2001, 61:474-477; Futaki et al., J. Biol. Chem., 2001,
February, 276(8):5836-5840; Cao G. et al., J. Neurosci., 2002,
22(13):5423-5431; Becker-Hapk, M. et al., Methods, 2001,
24:247-256; Snyder, E. L. and Dowdy, S. F., Curr. Opin. Mol. Ther.,
2001, 3:147-152; Lewin, M. et al., Nat. Biotechnol., 2000,
18:410-414; Tung, C. H. et al., Bioorg. Med. Chem., 2002,
10:3609-3614; Richard, J. P. , et al., J. Biol. Chem., Oct. 30,
2002, epub ahead of print). Transduction can occur in a receptor-
and transporter-independent fashion that appears to target the
lipid bilayer directly. Proteins (peptides) and compounds that are
linked to PTDs (e.g., covalently) have the capability to traverse
outer cell membranes. Preferably, the delivery peptide is a
trans-activating transcriptional activator (TAT) peptide or an
Antennapedia (ANT) peptide, or a derivative of either. PTDs can be
linked to the active agents for transport across the cell membrane.
One well characterized PTD is the human immunodeficient virus
(HIV)-1 Tat peptide (see, for example, U.S. Pat. Nos. 5,804,604;
5,747,641; 5,674,980; 5,670,617; and 5,652,122). Peptides such as
the homeodomain of Drosophila antennapedia (ANTP) and arginine-rich
peptides display similar properties can be employed. VP22, a
tegument protein from Herpes simplex virus type 1 (HSV-1), also has
the ability to transport proteins across a cell membrane, and may
be coupled to some PPIs.
Definitions
[0141] The terms "proximity probe" and "PLA probe" are used
interchangeably herein to refer to a moiety that binds to a target
molecule, such as a protein, in a sample and is detectable using
oligonucleotide amplification methods. In some embodiments, a
proximity probe comprises a target molecule recognition moiety and
an oligonucleotide probe. In some embodiments, a target molecule
specific amplification product can be formed when at least two
proximity probes specific for the target molecule are bound to the
target molecule and the oligonucleotide probe of each proximity
probe is ligated to one another to form a ligated probe that is
amplified.
[0142] Polymerase chain reaction (PCR) is a process for amplifying
one or more target nucleic acid sequences present in a nucleic acid
sample using primers and agents for polymerization and then
detecting the amplified sequence. The extension product of one
primer when hybridized to the other becomes a template for the
production of the desired specific nucleic acid sequence, and vice
versa, and the process is repeated as often as is necessary to
produce the desired amount of the sequence. The skilled artisan to
detect the presence of desired sequence (U.S. Pat. No. 4,683,195)
routinely uses polymerase chain reaction.
[0143] A specific example of PCR that is routinely performed by the
skilled artisan to detect desired sequences is reverse transcript
PCR (RT-PCR; Saiki et al., Science, 1985, 230:1350; Scharf et al.,
Science, 1986, 233:1076). RT-PCR involves isolating total RNA from
biological fluid, denaturing the RNA in the presence of primers
that recognize the desired nucleic acid sequence, using the primers
to generate a cDNA copy of the RNA by reverse transcription,
amplifying the cDNA by PCR using specific primers, and detecting
the amplified cDNA by electrophoresis or other methods known to the
skilled artisan.
[0144] As used herein, the terms "label" and "tag" refer to
substances that may confer a detectable signal (e.g., a signal
representing the amplification product of a proximity ligation
assay). A number of techniques for visualizing or detecting labeled
nucleic acids are readily available. Such techniques include,
microscopy, arrays, Fluorometry, Light cyclers or other real time
PCR machines, FACS analysis, scintillation counters,
Phosphoimagers, Geiger counters, MRI, CAT, antibody-based detection
methods (Westerns, immunofluorescence, immunohistochemistry),
histochemical techniques, HPLC, spectroscopy, capillary gel
electrophoresis, spectroscopy; mass spectroscopy; radiological
techniques; and mass balance techniques.
[0145] As used herein, the term "ligand" refers to a molecule that
contains a structural portion that is bound by specific interaction
with a particular receptor protein.
[0146] As used herein, the term "PPI" or "protein-protein
interaction" refers to refers to the binding of two or more
proteins together. PPIs may be binary (two protein binding
partners; a dimer) or tertiary (three or more protein binding
partners, e.g., a trimer). Proteins within a PPI (i.e., binding
partners) may be the same protein (such as a homodimer or
homotrimer) or different proteins (such as a heterodimer or
heterotrimer). Proteins within a tertiary interaction may be bound
to one or more proteins within the PPI. In some embodiments, the
PPI comprises a tyrosine kinase, such as the human tyrosine kinases
listed in Tables 3 and 4. The PPI may comprise an interaction
between the binding partners listed in Table 5 (human tyrosine
kinase interactions).
[0147] In some embodiments of the methods and kits of the
invention, at least one of the target binding partners is of an RTK
class selected from among RTK class I (EGF receptor family; ErbB
family), RTK class II (insulin receptor family), RTK class III
(PDGF receptor family), RTK class IV (FGF receptor family) RTK
class V (VEGF receptors family), RTK class VI (HGF receptor
family), RTK class VII (Trk receptor family), RTK class VIII (Eph
receptor family), RTK class IX (AXL receptor family), RTK class X
(LTK receptor family), RTK class XI (TIE receptor family), RTK
class XII (ROR receptor family), XIII (DDR receptor family), RTK
class XIV (RET receptor family), RTK class XV (KLG receptor
family), RTK class XVI (RYK receptor family), and RTK class XVII
(MuSK receptor family).
[0148] As used herein, the term "PPI profile" or "protein-protein
interaction profile" refers to the result or output of an assay
that measures the relative abundance of a protein-protein
interaction. PPI profiles (e.g., sample PPI profile, and reference
PPI profile) may each be expressed as a value representative of the
abundance of target binding partners in proximity to each other
within the sample. The sample PPI profile and reference PPI profile
may be expressed by any method useful for comparison purposes, such
as a numeric value, score, cutoff (threshold), or other expression.
For example, a negative result in which a PPI profile in a sample
does not reach the cutoff would be useful clinically to avoid
giving patients unnecessary PPI modulator therapy. A positive
result in which a PPI profile in a sample is at or above the cutoff
could indicate potential sensitivity and allow the clinician to
give the PPI modulator therapy to those patients who would be most
likely to benefit. Typically, a reference PPI profile is the PPI
profile of a known cancer cell (e.g., cancer cell of a known type
or subtype) or of a normal cell (non-cancerous cell) useful for
comparison purposes. The reference PPI profile may also be a sample
PPI profile obtained at a different time point than the sample PPI
profile in question (before or after, to observe changes to the PPI
profile of a cancer over time).
[0149] As used herein, the term "PPI modulator" refers to an agent
(e.g., small molecule, protein, nucleic acid) that directly or
indirectly promotes, inhibits, or otherwise alters interaction
between two or more proteins. The PPI modulator may be an inducer
of the PPI (directly or indirectly promoting interaction), or an
inhibitor of the PPI (directly or indirectly inhibiting
interaction). For example, a PPI inhibitor may be a kinase
inhibitor, which indirectly acts to inhibit PPI.
[0150] As used herein, the term "bind" refers to any physical
attachment or close association, which may be permanent or
temporary. The binding can result from hydrogen bonding,
hydrophobic forces, van der Waals forces, covalent, or ionic
bonding, for example.
[0151] As used herein, the term "antibody" refers to immunoglobulin
molecules and immunologically active portions (fragments) of
immunoglobulin molecules, i.e., molecules that contain an antibody
combining site or paratope. The term is inclusive of monoclonal
antibodies and polyclonal antibodies.
[0152] As used herein, the terms "administering" or "administer"
are used herein to refer the introduction of a substance into cells
in vitro or into the body of an individual in vivo by any route
(for example, oral, nasal, ocular, rectal, vaginal and parenteral
routes). Active agents such as PPI modulators may be administered
individually or in combination with other agents via any route of
administration, including but not limited to subcutaneous (SQ),
intramuscular (IM), intravenous (IV), intraperitoneal (IP),
intradermal (ID), via the nasal, ocular or oral mucosa (IN), or
orally. For example, active agents such as PPI modulators can be
administered by direct injection into or on a tumor, or
systemically (e.g., into the circulatory system), to kill
circulating tumor cells (CTC).
[0153] As used herein, the term "polypeptide" refers to a sequence
of two or more amino acids, and is used interchangeably herein with
the terms "protein", "gene product", "oligopeptide", and
"peptide".
[0154] In the context of the instant invention, the terms
"oligopeptide", "polypeptide", "peptide" and "protein" can be used
interchangeably to refer to amino acid sequences of any length;
however, it should be understood that the invention does not relate
to the peptides in natural form, that is to say that they are not
in their natural environment but that the peptide may have been
isolated or obtained by purification from natural sources or
obtained from host cells prepared by genetic manipulation (e.g.,
the peptides, or fragments thereof, are recombinantly produced by
host cells, or by chemical synthesis). Peptide PPI modulators may
also contain non-natural amino acids, as will be described below.
The terms "oligopeptide", "polypeptide", "peptide" and "protein"
are also used, in the instant specification, to designate a series
of residues of any length, typically L-amino acids, connected one
to the other, typically by peptide bonds between the a-amino and
carboxyl groups of adjacent amino acids. Linker elements can be
joined to the peptides, for example, through peptide bonds or via
chemical bonds (e.g., heterobifunctional chemical linker elements)
as set forth below. Additionally, the terms "amino acid(s)" and
"residue(s)" can be used interchangeably.
[0155] As used herein, the terms "treat" or "treatment" refer to
both therapeutic treatment and prophylactic or preventative
measures, wherein the object is to prevent or slow down (lessen) an
undesired physiological change or disorder, such as the development
or spread of cancer or other proliferation disorder. For purposes
of this invention, beneficial or desired clinical results include,
but are not limited to, alleviation of symptoms, diminishment of
extent of disease, stabilized (i.e., not worsening) state of
disease, delay or slowing of disease progression, amelioration or
palliation of the disease state, and remission (whether partial or
total), whether detectable or undetectable. For example, treatment
with active agent such as a PPI modulator may include reduction of
undesirable cell proliferation, and/or induction of apoptosis and
cytotoxicity. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment. Those in
need of treatment include those already with the condition or
disorder as well as those prone to have the condition or disorder
or those in which the condition or disorder is to be prevented or
onset delayed. Optionally, the patient may be identified (e.g.,
diagnosed) as one suffering from the disease or condition (e.g.,
proliferation disorder) prior to administration of the PPI
modulator.
[0156] As used herein, the term "(therapeutically) effective
amount" refers to an amount of the PPI modulator or other active
agent (e.g., a drug) effective to treat a disease or disorder in a
mammal. In the case of cancer or other proliferation disorder, the
therapeutically effective amount of the agent may reduce (i.e.,
slow to some extent and preferably stop) unwanted cellular
proliferation; reduce the number of cancer cells; reduce the tumor
size; inhibit (i.e., slow to some extent and preferably stop)
cancer cell infiltration into peripheral organs; inhibit (i.e.,
slow to some extent and preferably stop) tumor metastasis; inhibit,
to some extent, tumor growth; reduce signaling in the target cells,
and/or relieve, to some extent, one or more of the symptoms
associated with the cancer. To the extent the administered PPI
modulator prevents growth of and/or kills existing cancer cells, it
may be cytostatic and/or cytotoxic. For cancer therapy, efficacy
can, for example, be measured by assessing the time to disease
progression (TTP) and/or determining the response rate (RR).
[0157] Treatments can be given in a growth inhibitory amount. As
used herein, the term "growth inhibitory amount" of the active
agent (e.g., PPI modulator) refers to an amount which inhibits
growth or proliferation of a target cell, such as a tumor cell,
either in vitro or in vivo, irrespective of the mechanism by which
cell growth is inhibited (e.g., by cytostatic properties, cytotoxic
properties, etc.). In a preferred embodiment, the growth inhibitory
amount inhibits (i.e., slows to some extent and preferably stops)
proliferation or growth of the target cell in vivo or in cell
culture by greater than about 20%, preferably greater than about
50%, most preferably greater than about 75% (e.g., from about 75%
to about 100%).
[0158] The terms "cell" and "cells" are used interchangeably herein
and are intended to include either a single cell or a plurality of
cells, in vitro or in vivo, unless otherwise indicated.
[0159] As used herein, the term "sample" refers a biological
composition that potentially contains target molecules (e.g., one
or more protein binding partners in a protein-protein interaction
of interest). Preferably, the sample is a cellular sample (samples
of intact cells, e.g., a cytology sample). One or more samples of a
malignancy may be obtained from a subject by techniques known in
the art, such as biopsy. The type of biopsy utilized is dependent
upon the anatomical location from which the sample is to be
obtained. Examples include fine needle aspiration (FSA), excisional
biopsy, incisional biopsy, colonoscopic biopsy, punch biopsy, and
bone marrow biopsy. Depending on the PPI of interest, the sample
may be obtained from a pleural effusion or the bloodstream, for
example. It should be understood that the methods of the invention
may include a step in which a sample is obtained directly from a
subject; alternatively, a sample may be obtained or otherwise
provided, e.g., by a third party.
[0160] A sample may be taken from a subject having or suspected of
having cancer. A sample may also comprise proteins isolated from a
tissue or cell sample from a subject. In certain aspects, the
sample can be, but is not limited to tissue (e.g., biopsy,
particularly fine needle biopsy, excision, or punch biopsy), blood,
serum, plasma. The sample can be fresh, frozen, fixed (e.g.,
formalin fixed), or embedded (e.g., paraffin embedded) tissues or
cells (e.g., FFPE tissue). In a particular aspect, the sample is a
sample of lung cancer cells, colon cancer cells, breast cancer
cells, ovarian cancer cells, renal cancer cells, melanoma cells,
prostate cancer cells, CNS cancer cells, or leukemia cells,
esophageal cancer cells, stomach cancer cells, bile duct cancer
cells, liver cancer cells, cancer cells of the rectum or anus,
lymphoma, leukemia, cervical cancer cells, bladder cancer cells, or
protein from any of the aforementioned cancers. Depending upon the
cancer type, the cancer cells may be circulating tumor cells
(CTCs).
[0161] As used herein, the term "anti-cancer agent" refers to a
substance or treatment (e.g., agent or radiation therapy) that
inhibits the function of cancer cells, inhibits their growth,
formation, and/or causes their destruction in vitro or in vivo.
Examples include, but are not limited to, cytotoxic agents (e.g.,
5-fluorouracil, TAXOL), chemotherapeutic agents, and anti-signaling
agents (e.g., the PI3K inhibitor LY). Anti-cancer agents include
but are not limited to the chemotherapeutic agents listed in Table
2, and other agents disclosed herein, such as erlotinib or other
small molecule epidermal growth factor tyrosine kinase inhibitors
(EGFR TKI). In the kits and methods of the invention, the treatment
or potential treatment may be an anti-cancer agent (for example, a
PPI modulator such as a kinase inhibitor or other PPI
inhibitor).
[0162] As used herein, the term "cytotoxic agent" refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells in vitro and/or in vivo. The term is
intended to include radioactive isotopes (e.g., At.sup.211,
I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, and radioactive isotopes of Lu),
chemotherapeutic agents, toxins such as small molecule toxins or
enzymatically active toxins of bacterial, fungal, plant or animal
origin, and antibodies, including fragments and/or variants
thereof.
[0163] As used herein, the term "chemotherapeutic agent" is a
chemical compound useful in the treatment of cancer, such as, for
example, taxanes, e.g., paclitaxel (TAXOL, BRISTOL-MYERS SQUIBB
Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE, Rhone-Poulenc
Rorer, Antony, France), chlorambucil, vincristine, vinblastine,
anti-estrogens including for example tamoxifen, raloxifene,
aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY117018, onapristone, and toremifene
(FARESTON, GTx, Memphis, Tenn.), and anti-androgens such as
flutamide, nilutamide, bicalutamide, leuprolide, and goserelin,
etc. Examples of chemotherapeutic agents that may be used in
conjunction with PPI modulators are listed in Table 2. In a
preferred embodiment, the chemotherapeutic agent is one or more
anthracyclines. Anthracyclines are a family of chemotherapy drugs
that are also antibiotics. The anthracyclines act to prevent cell
division by disrupting the structure of the DNA and terminate its
function by: (1) intercalating into the base pairs in the DNA minor
grooves; and (2) causing free radical damage of the ribose in the
DNA. The anthracyclines are frequently used in leukemia therapy.
Examples of anthracyclines include daunorubicin (CERUBIDINE),
doxorubi cin (ADRIAMYCIN, RUBEX), epirubicin (ELLENCE,
PHARMORUBICIN), and idarubicin (IDAMYCIN).
TABLE-US-00002 TABLE 2 Examples of Chemotherapeutic Agents
13-cis-Retinoic Acid Mylocel 2-Amino-6- Letrozole Mercaptopurine
Neosar 2-CdA Neulasta 2-Chlorodeoxyadenosine Neumega 5-fluorouracil
Neupogen 5-FU Nilandron 6-TG Nilutamide 6-Thioguanine Nitrogen
Mustard 6-Mercaptopurine Novaldex 6-MP Novantrone Accutane
Octreotide Actinomycin-D Octreotide acetate Adriamycin Oncospar
Adrucil Oncovin Agrylin Ontak Ala-Cort Onxal Aldesleukin Oprevelkin
Alemtuzumab Orapred Alitretinoin Orasone Alkaban-AQ Oxaliplatin
Alkeran Paclitaxel All-transretinoic acid Pamidronate Alpha
interferon Panretin Altretamine Paraplatin Amethopterin Pediapred
Amifostine PEG Interferon Aminoglutethimide Pegaspargase Anagrelide
Pegfilgrastim Anandron PEG-INTRON Anastrozole PEG-L-asparaginase
Arabinosylcytosine Phenylalanine Mustard Ara-C Platinol Aranesp
Platinol-AQ Aredia Prednisolone Arimidex Prednisone Aromasin
Prelone Arsenic trioxide Procarbazine Asparaginase PROCRIT ATRA
Proleukin Avastin Prolifeprospan 20 with Carmustine implant BCG
Purinethol BCNU Raloxifene Bevacizumab Rheumatrex Bexarotene
Rituxan Bicalutamide Rituximab BiCNU Roveron-A (interferon alfa-2a)
Blenoxane Rubex Bleomycin Rubidomycin hydrochloride Bortezomib
Sandostatin Busulfan Sandostatin LAR Busulfex Sargramostim C225
Solu-Cortef Calcium Leucovorin Solu-Medrol Campath STI-571
Camptosar Streptozocin Camptothecin-11 Tamoxifen Capecitabine
Targretin Carac Taxol Carboplatin Taxotere Carmustine Temodar
Carmustine wafer Temozolomide Casodex Teniposide CCNU TESPA CDDP
Thalidomide CeeNU Thalomid Cerubidine TheraCys cetuximab
Thioguanine Chlorambucil Thioguanine Tabloid Cisplatin
Thiophosphoamide Citrovorum Factor Thioplex Cladribine Thiotepa
Cortisone TICE Cosmegen Toposar CPT-11 Topotecan Cyclophosphamide
Toremifene Cytadren Trastuzumab Cytarabine Tretinoin Cytarabine
liposomal Trexall Cytosar-U Trisenox Cytoxan TSPA Dacarbazine VCR
Dactinomycin Velban Darbepoetin alfa Velcade Daunomycin VePesid
Daunorubicin Vesanoid Daunorubicin Viadur hydrochloride Vinblastine
Daunorubicin liposomal Vinblastine Sulfate DaunoXome Vincasar Pfs
Decadron Vincristine Delta-Cortef Vinorelbine Deltasone Vinorelbine
tartrate Denileukin diftitox VLB DepoCyt VP-16 Dexamethasone Vumon
Dexamethasone acetate Xeloda dexamethasone sodium Zanosar phosphate
Zevalin Dexasone Zinecard Dexrazoxane Zoladex DHAD Zoledronic acid
DIC Zometa Diodex Gliadel wafer Docetaxel Glivec Doxil GM-CSF
Doxorubicin Goserelin Doxorubicin liposomal granulocyte - colony
stimulating factor Droxia Granulocyte macrophage colony stimulating
DTIC factor DTIC-Dome Halotestin Duralone Herceptin Efudex Hexadrol
Eligard Hexalen Ellence Hexamethylmelamine Eloxatin HMM Elspar
Hycamtin Emcyt Hydrea Epirubicin Hydrocort Acetate Epoetin alfa
Hydrocortisone Erbitux Hydrocortisone sodium phosphate Erwinia
L-asparaginase Hydrocortisone sodium succinate Estramustine
Hydrocortone phosphate Ethyol Hydroxyurea Etopophos Ibritumomab
Etoposide Ibritumomab Tiuxetan Etoposide phosphate Idamycin Eulexin
Idarubicin Evista Ifex Exemestane IFN-alpha Fareston Ifosfamide
Faslodex IL-2 Femara IL-11 Filgrastim Imatinib mesylate Floxuridine
Imidazole Carboxamide Fludara Interferon alfa Fludarabine
Interferon Alfa-2b (PEG conjugate) Fluoroplex Interleukin-2
Fluorouracil Interleukin-11 Fluorouracil (cream) Intron A
(interferon alfa-2b) Fluoxymesterone Leucovorin Flutamide Leukeran
Folinic Acid Leukine FUDR Leuprolide Fulvestrant Leurocristine
G-CSF Leustatin Gefitinib Liposomal Ara-C Gemcitabine Liquid Pred
Gemtuzumab ozogamicin Lomustine Gemzar L-PAM Gleevec L-Sarcolysin
Lupron Meticorten Lupron Depot Mitomycin Matulane Mitomycin-C
Maxidex Mitoxantrone Mechlorethamine M-Prednisol Mechlorethamine
MTC Hydrochlorine MTX Medralone Mustargen Medrol Mustine Megace
Mutamycin Megestrol Myleran Megestrol Acetate Iressa Melphalan
Irinotecan Mercaptopurine Isotretinoin Mesna Kidrolase Mesnex
Lanacort Methotrexate L-asparaginase Methotrexate Sodium LCR
Methylprednisolone
[0164] As used herein, the term "tumor" refers to all neoplastic
cell growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. For example, a
particular cancer may be characterized by a solid tumor mass. A
primary tumor mass refers to a growth of cancer cells in a tissue
resulting from the transformation of a normal cell of that tissue.
In most cases, the primary tumor mass is identified by the presence
of a cyst, which can be found through visual or palpation methods,
or by irregularity in shape, texture, or weight of the tissue.
However, some primary tumors are not palpable and can be detected
only through medical imaging tec;hniques such as X-rays (e.g.,
mammography), or by needle aspirations. The use of these latter
techniques is more common in early detection. Molecular and
phenotypic analysis of cancer cells within a tissue will usually
confirm if the cancer is endogenous to the tissue or if the lesion
is due to metastasis from another site. The peptide PPI modulators
may be capable of inducing apoptosis in tumor cells, reducing tumor
size, and/or inhibiting tumor cell growth. The peptides of the
invention (or nucleic acids encoding them) can be administered
locally at the site of a tumor (e.g., by direct injection) or
remotely. The peptide PPI modulators may induce cell death in
circulating tumor cells (CTC) in a subject, e.g., by administering
the peptides or encoding nucleic acids intravenously. Furthermore,
the peptide PPI modulators may prevent or reduce onset of
metastasis to other tissues, e.g., to the bone.
[0165] As used herein, the term "signaling" and "signaling
transduction" represents the biochemical process involving
transmission of extracellular stimuli, via cell surface receptors
through a specific and sequential series of molecules, to genes in
the nucleus resulting in specific cellular responses to the
stimuli.
[0166] As used herein, the term "pharmaceutically acceptable salt
or prodrug" is intended to describe any pharmaceutically acceptable
form (such as an ester, phosphate ester, salt of an ester or a
related group) of a PPI modulator or other active agent, which,
upon administration to a subject, provides the mature or base
compound. Pharmaceutically acceptable salts include those derived
from pharmaceutically acceptable inorganic or organic bases and
acids. Suitable salts include those derived from alkali metals such
as potassium and sodium, alkaline earth metals such as calcium and
magnesium, among numerous other acids well known in the
pharmaceutical art. Pharmaceutically acceptable prodrugs refer to a
compound that is metabolized, for example hydrolyzed or oxidized,
in the host to form the compound of the present invention. Typical
examples of prodrugs include compounds that have biologically
labile protecting groups on a functional moiety of the active
compound. Prodrugs include compounds that can be oxidized, reduced,
aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed,
dehydrolyzed, alkylated, dealkylated, acylated, deacylated,
phosphorylated, dephosphorylated to produce the active
compound.
[0167] The terms "link" or "join" refers to any method known in the
art for functionally connecting peptides, including, without
limitation, recombinant fusion, covalent bonding, disulfide
bonding, ionic bonding, hydrogen bonding, and electrostatic
bonding.
[0168] The terms "comprising", "consisting of and "consisting
essentially of are defined according to their standard meaning. The
terms may be substituted for one another throughout the instant
application in order to attach the specific meaning associated with
each term.
[0169] The terms "isolated" or "biologically pure" refer to
material that is substantially or essentially free from components
which normally accompany the material as it is found in its native
state.
[0170] As used in this specification, the singular forms "a", "an",
and "the" include plural reference unless the context clearly
dictates otherwise. Thus, for example, a reference to "a compound"
includes more than one such compound. Reference to "PPI modulator"
includes more than one such PPI modulator. A reference to "an
antibody" includes more than one such antibody, and so forth.
[0171] The practice of the present invention can employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA technology, electrophysiology, and
pharmacology that are within the skill of the art. Such techniques
are explained fully in the literature (see, e.g., Sambrook, Fritsch
& Maniatis, Molecular Cloning: A Laboratory Manual, Second
Edition (1989); DNA Cloning, Vols. I and II (D. N. Glover Ed.
1985); Perbal, B., A Practical Guide to Molecular Cloning (1984);
the series, Methods In Enzymology (S. Colowick and N. Kaplan Eds.,
Academic Press, Inc.); Transcription and Translation (Hames et al.
Eds. 1984); Gene Transfer Vectors For Mammalian Cells (J. H. Miller
et al. Eds. (1987) Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y.); Scopes, Protein Purification: Principles and
Practice (2nd ed., Springer-Verlag); and PCR: A Practical Approach
(McPherson et al. Eds. (1991) IRL Press)), each of which are
incorporated herein by reference in their entirety.
[0172] Experimental controls are considered fundamental in
experiments designed in accordance with the scientific method. It
is routine in the art to use experimental controls in scientific
experiments to prevent factors other than those being studied from
affecting the outcome.
Materials and Methods
[0173] Primary Xenograft Mouse Models. 4-6 weeks old female
hairless mice Crl:SHO-Prkdc-SCID-Hr-hr mice will be purchased from
approved vendors and housed in the institutional animal facilities
according to protocols set out by the American Association for
Accreditation of Laboratory Animal Care. The inventor has active
IACUC protocols for establishing tumor xenografts in mice and to
perform drug treatment experiments. Mice will be anesthetized with
isofluorane, a small incision and a small subcutaneous pocket will
be made in each side of the lower back, and a piece of tumor
collected from NSCLC patients will be deposited in each pocket.
Animals will be observed until palpable tumors are present and
subsequently treated vehicle (31). The mice will be monitored 2-3
times per week. If any mice demonstrate discomfort, i.e., lethargy,
abnormal posture, failure to groom, ruffled, matted or soiled hair
coat, rapid, shallow and/or labored breathing, have tumors greater
than 1.0 cm in diameter, have tumors which interfere with posture,
locomotion or feeding, or have tumors abscess through the skin,
they will be euthanatized using CO.sub.2. Each group of mice will
be put in the cleaned and dried chamber, and then the chamber will
be slowly filled by compressed carbon dioxide gas. Mice will be
quickly euthanatized by carbon dioxide inhalation. Mouse death will
be confirmed by verification of cessation of cardiovascular and
respiratory movement/activity. This method is consistent with
recommendations of the Panel on Euthanasia of the American
Veterinary Medical Association. Log entries for complications of
either tumor growth or tumor metastasis will be maintained. For
tumor harvesting, mice will be euthanized by CO.sub.2 inhalation
and tumors will be excised and snap frozen in liquid nitrogen.
[0174] Cell Line Xenograft Models. Tumor tissues from subcutaneous
mouse models of lung cancer will be used. CD-1 nu/nu mice do not
have a thymus and are therefore immunodeficient and unable to
produce T-cells. Mice have been selected for these experiments
since they are most suitable for xenograft and orthographic studies
of lung cancer, are readily available and not endangered, and can
be safely maintained in the laboratory animal vivarium.CD-1 nu/nu
mice mice will be purchased from approved vendors and housed in
institutional animal facilities according to protocols set out by
the American Association for Accreditation of Laboratory Animal
Care. The inventor currently has active IACUC protocol for
establishing subcutaneous tumor xenografts in mice. Both male and
female mice of approximately six weeks of age will be used. Mice
will be anesthetized in an induction chamber containing 3%
isoflurane and 97% oxygen. Mice will be subsequently shaved in the
flank area and injected with various human lung cancer cell lines.
Animals will be observed until palpable tumors are present and
subsequently treated vehicle or compounds in 0.2 ml vehicle
containing 0.5% methylcellulose and 0.4% polysorbate 80 (TWEEN 80)
(59, 60). The mice will be monitored 2-3 times per week. If any
mice demonstrate discomfort, i.e., lethargy, abnormal posture,
failure to groom, ruffled, matted or soiled hair coat, rapid,
shallow and/or labored breathing, have tumors greater than 1.0 cm
in diameter, have tumors which interfere with posture, locomotion
or feeding, or have tumors abscess through the skin, they will be
euthanatized using CO2. Each group of mice will be put in the
cleaned and dried chamber, and then the chamber will be slowly
filled by compressed carbon dioxide gas. Mice will be quickly
euthanatized by carbon dioxide inhalation. Mouse death will be
confirmed by verification of cessation of cardiovascular and
respiratory movement/activity. This method is consistent with
recommendations of the Panel on Euthanasia of the American
Veterinary Medical Association. Log entries for complications of
either tumor growth or tumor metastasis will be maintained. For
tumor harvesting, mice will be euthanized by CO2 inhalation and
tumors will be excised and snap frozen in liquid nitrogen. [0175]
Patient consenting process: The TCC protocol was IRB approved in
January 2006; since then, 33,000+ patients have prospectively
consented to have their tissue collected and agreed to lifetime
follow-up. Moffitt expects 100,000 consents within 4 years at 15
partner institutions. [0176] Sample Collection: To date, over
10,000 tumors have been collected. This number will grow
exponentially in coming years. TCC collects tumor, blood and urine
samples as well as clinical data such as risk factors, therapies
and outcomes. All tissues are snap frozen within 15 minutes of
resection and 2D bar code labeled. Moffitt has robotic biobanking
and every tissue is macrodissected to .gtoreq.85% purity. [0177]
Pathologic Review of Frozen Tissue Specimens: Each frozen tissue
specimen is subjected to comprehensive histopathologic review by
Board-certified Pathologists. Each tumor sample is quantified for
proportions of the following tissue components: malignant (viable
tumor cellularity, tumor necrosis and stroma), normal, abnormal
(including inflammation, ulceration, atrophy, hyperplasia etc) and
benign neoplastic tissues. In order to achieve the highest possible
purity of viable malignant tumor tissue for each specimen, every
effort is made to exclude (macrodissect) the latter three
components from the tissue processed for subsequent molecular
analyses. In order to ensure the highest quality of tissue
specimens and to control for pre-analytic variables, the tissue
collection and review processes are regulated by specific Standard
Operating Procedures (SOPs) and QA/QC protocols. [0178] Gene
Expression Profiling: 7,000 samples have been analyzed to date.
Identifying molecular signatures is a key component of
precision-based personalized medicine. To date, TCC has focused on
gene expression but other modalities, including quantitative
proteomics, are planned. [0179] Biorepository: All specimens
collected under the TCC protocol are stored in a single location at
Moffitt in a biobank capable of storing 1,000,000 samples.
Currently, tumor specimens are collected from patients with colon,
lung, prostate, brain, breast, pancreatic, ovarian, bladder, and
renal cancers. As the resource develops, tumor specimens from all
disease sites will be acquired from consortium members. [0180] Data
Warehouse: Storage is provided for patient records, medical
history, registry data, molecular profiles (e.g. gene expression),
and clinicopathologic data in compliance with HIPAA requirements.
Within 5 years, the warehouse is expected to hold information on
primary and metastatic biopsies for over 50,000 patients.
Appropriate access is given to the patient, the clinician and
researcher to lead to the creation of evidence-based guidelines.
Separate web portals are being developed to meet these distinct
needs.
[0181] Translational research is incorporated along this continuum
of care and follow-up. TCC is more than just a registry, cohort
study, or tissue bank; the system prospectively consents patients,
collects clinical data throughout a patient's lifetime, creates
gene expression profiles of all tumors, and enables access to the
data for the patient, clinician and researcher. Its capability for
in-depth analysis could lead to improving the standard of care
addressing each patient's specific needs.
[0182] Subjects are being recruited to Total Cancer Care.TM. (TCC)
sites in FL (including the Moffitt Cancer Center (MCC), primary
sponsor of the study), NC, SC, CT, KY, LA, IN and NE according to
the TCC protocol and consent. The total recruitment expected across
all sites is by 2012 is 100,000 patients. Patients ages 18 and
older years with newly diagnosed, recurrent or metastatic cancers
(stages I-IV) are being recuited. Inclusion criteria are broad to
include any willing patient that has visited one of the consortium
sites and presented with a cancer diagnosis. Exclusion criteria is
any registered patient who is not willing or able to sign the
consent for data collection. The TCC study provides for the
collection of patient biospecimens and data throughout the patients
life in a large prospective study. Tumor samples collected either
through resection or biopsy for research studies are comprised of
excess tissue not necessary for pathological diagnosis or clinical
care and are banked by MCC. No appreciable risks to subjects
associated with tissue collection and analysis is anticipated; the
patient's surgeon will remove this tissue as part of his or her
clinical care, and no additional tissue will be removed for the TCC
Study unless specified and consent granted by the patient prior to
the procedure. Additional biospecimens, including blood and urine,
maybe collected as described in the TCC protocol and consent
documents, although these are not used for the study purposes of
the proposed research described in this application. If there is a
change in protocol procedures or desired biospecimen types an
amendment to the protocol and consent are provided to the
University of South Florida's Internal Review Board (IRB) for
consideration of the benefits to research and the impact on patient
care, well being, harm and discomfort. Changes to the TCC protocol
at Consortium sites are presented to local IRBs for consideration
prior to additional biospecimen collections. Subjects may feel
uncomfortable answering personal questions on study questionnaires
or knowing that their medical records are being reviewed. Patients
will be informed that that they may skip any questions they prefer
not to answer. Great care will be taken to protect patient privacy
throughout the study in accordance with MCC policies.
[0183] The invenor has an active protocol that also allows for
patient chart review to gather information necessary for this
project as well as access tumor tissue for molecular assays. This
study was approved by the University of South Florida IRB and
allows access to PHI for research purposes described in this
proposal. This allows for chart review by the inventor and his
trained staff to identify clinical variables such as age, sex,
histology of tumor, stage, response to therapy, and overall
survival.
[0184] Some of the Figures herein have been labeled with one or
more colors to facilitate interpretation when the Figures are
viewed in black and white. The lack of a color label on any
particular region of a figure should not necessarily be construed
as an absence of color in that region.
[0185] All patents, patent applications, provisional applications,
and publications referred to or cited herein, supra or infra, are
incorporated by reference in their entirety, including all figures
and tables, to the extent they are not inconsistent with the
explicit teachings of this specification.
[0186] Following are examples which illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
EXAMPLE 1
Epidermal Growth Factor Receptor (egfr) Protein-Protein
Interactions as Biomarkers
[0187] Cancer is recognized to be a result of changes in cellular
genomes resulting in aberrant signaling proteins causing
deregulated cell growth, survival, and metastasis. These changes
rewire entire signaling `circuits` resulting in aberrant growth and
metastasis. Critical to protein function and signaling is the
formation of signaling complexes and networks of signaling proteins
that act in concert to produce a physiological signal (1, 2). State
of the art mass spectrometry is now able to accurately map
protein-protein interaction (PPI) complexes and networks (3, 4).
Initially, this was performed in yeast with high impact papers
demonstrating the modular organization of PPI and insight into
cellular signaling mechanisms (5, 6). However, improvements in mass
spectrometry coupled with improved biochemical purification
strategies are now enabling PPI mapping in mammalian cells,
including cancer cells (7-9). This allows a better understanding of
how cancer proteins drive a signaling network to transform cells.
The application of network theory to biology may enable a better
understanding of cancer, improve ability to classify tumors, and
suggest therapeutic approaches against cancer `hub` proteins or
suggest rational combination approaches (10-14). In addition,
comprehensive databases (such as www.hprd.org) now list protein
interactions based on manually annotated reviews of the literature.
Using these databases, groups can construct theoretical PPI that
could be important in diseases such as cancer. One group used these
databases to demonstrate the importance of deregulated PPI in
aggressive cancers (15). Importantly, this network view can produce
biomarkers categorized by protein-protein interactions that carry
information to guide clinical decision making. Despite the
explosion of PPI datasets, most are limited in pre-clinical space
and interrogations of PPI in human cancer specimens is lacking.
Thus, the present inventors have endeavored to make `network
medicine` a reality (16).
[0188] The present inventors have initiated a system wide and
global analysis of the PPI driven by hyperactivated epidermal
growth factor receptor (EGFR) resulting from somatic mutation in
non-small cell lung cancer (NSCLC) (17-19). The inventors' approach
has been to combine (i) affinity purification--mass spectrometry
(AP-MS) and (ii) phosphotyrosine proteomics. The inventors use cell
models that harbor mutant EGFR proteins and are highly sensitive to
EGFR TKI. As the network of EGFR is highly dependent on cell
context, this approach offers the best chance of recovering
pertinent proteins and mechanisms, a potential flaw in using cells
other than lung cancer cells driven by mutant EGFR. Tagged versions
of EGFR are initially used to identify interactions and second
round of AP-MS is performed to more fully construct the network
(20, 21). The inventors simultaneously performed tyrosine
phosphoproteomics--tyrosine phosphorylated (pTyr) peptides are
enriched using an anti-phosphotyrosine antibody and then identified
and quantified on an Orbitrap mass spectrometer using published
methods (9). Using protein-protein interaction databases, the
inventors linked pTyr containing proteins to proteins identified in
AP-MS experiments. This produced the final map of nearly 300
proteins (shown in FIGS. 1A-1D). This interaction network allows
functional interrogation of targets important in driving mutant
EGFR growth and survival signals. In addition, the interactions
themselves can be used as biomarkers in human cancer specimens by
identifying an EGFR activating state. Thus, the critical question
becomes, how can one `translate` these preclinical mass
spectrometry data into human cancer samples?
[0189] Translation of these network approaches to tumor samples is
hampered by a number of challenges. First, almost all studies have
been carried out in engineered mammalian cells that express a
tagged version of the protein of interest; this limits the ability
to use these modern AP-MS approaches in samples from patients.
Second, most samples from patients are formalin fixed and paraffin
embedded. This precludes the ability to use fresh frozen tissue for
immunoprecipitation and western blotting (IP-WB) to identify
protein complexes. IP-WB also requires a large amount of starting
material that can be difficult to obtain with needle biopsies.
[0190] One solution to mapping networks identified using MS-based
proteomics is proximity ligation assays (PLA) (22-27). Briefly, two
proteins in complex are each identified with primary antibodies
specific for the protein and linked to a conjugated
oligonucleotide. A connector oligonucleotide links both proximity
probes allowing ligation and formation of a template for PCR
amplification. The resulting rolling circular amplification (RCA)
serves as a target for hybridized fluorescently labeled detection
oligonucleotides allowing distinct and bright spots to be
identified and quantified in a fluorescent microscope. See
http://www.olink.webbhuset.com/movie.php for an animation
describing this technology. This technology has been used to
identify in cells in situ Myc-Max interactions, tyrosine
phosphorylation of receptor tyrosine kinases, and interactions of
proteins in human tissue sections.
[0191] As little has been done to establish biomarker systems to
measure protein-protein based biomarkers in cancer, it would be
advantageous to have an assay (e.g., a PLA) that can quantitatively
measure defined protein-protein interactions in cancer specimens
(e.g., EGFR protein-protein interactions in lung cancer specimens)
and relate expression of these interactions to clinical outcome
variables. Lung cancer accounts for over 160,000 deaths per year in
the U.S., more than breast, colon, prostate and pancreatic cancer
combined (28). The overall five-year survival rate for lung cancer
is approximately 15%, and unlike other solid tumors such as colon
or breast cancer, little progress has been made in improving
survival. There is therefore an important unmet need to better
identify key drivers of lung cancer that can be therapeutically
exploited. Coupled with this need is the ability to identify
patients' subsets driven by drug targets. EGFR is selected as a
test case to develop these assays based on (i) its known role in
lung cancer pathogenesis, (ii) use of EGFR TKI for treatment of
lung cancer, (iii) experimentally derived protein-protein
interaction data derived in the inventor's laboratory using mass
spectrometry and (iv) availability of antibody reagents (29). The
assays are not meant to replace existing clinical assays that
predict EGFR kinase inhibitor sensitivity (i.e., EGFR mutation
analysis) but are meant to allow a starting point to develop PPI
based biomarkers that could be applied to other cancer pathways or
drugs. This could allow markers that identify patients benefiting
from EGFR TKI whose tumors do not harbor EGFR mutations. Finally,
success here can be translated to other signaling systems, such as
MET and IGFR, where interactions could be informative (for example,
MET with Gab 1 and IGFR with IRS proteins) and biomarkers
predicting response are unclear (30).
[0192] The approach to develop biomarkers based on protein-protein
interactions goes one step beyond measurement of protein expression
as it determines the binding to two proteins together in tumor
tissues. Two proteins may be equally expressed, but because of
other nuances in the cancer cell, do not form a signaling complex
that drives a signaling cascade. In another tumor cell, these
proteins form a complex and drive a signaling cascade. Gene-based
approaches that measure mRNA expression or immunohistochemistry
approaches that measure protein expression would be unable to
discriminate these two cases. One aspect of the invention is a
translatable assay system measuring protein-protein interactions
driven by hyperactivated forms of EGFR and this approach will be
tested in panels of cell lines, animal tumor models, and specimens
from lung cancer patients. The inventor builds on the AP-MS mass
spectrometry data to build protein-protein interaction networks in
lung cancer cell lines driven by activated EGFR and translate these
findings into patient based materials. Four aims are described
herein.
[0193] Aim 1: Establish and validate proximity ligation assays that
measure EGFR protein-protein interactions
[0194] The invention provides assays measuring protein-protein
interactions based on proximity ligation assay (PLA) technology.
This technology is capable of detecting single protein events such
as protein interactions. The assay provides exact spatial
information on the location of the events and an objective means of
quantifying the events. Experimentally derived mass spectrometry
data defining interacting proteins within the EGFR network will
guide selection of protein complexes for assay development.
[0195] Aim 2: Characterize EGFR protein interactions in cell and
tumor models with known EGFR mutation status. Here the hypothesis
is that cells and tumors with activating EGFR mutations will
demonstrate constitutive complexes with EGFR proteins. This will be
tested in both cell lines with known EGFR mutation status and tumor
samples from patients with known EGFR mutation status. The inventor
will also test the hypothesis that the degree of EGFR complexes is
related to sensitivity to EGFR tyrosine kinase inhibitors
(TKI).
[0196] Aim 3: Characterize changes in EGFR protein-protein
interactions in response to EGFR tyrosine kinase inhibitors in
primary lung cancer xenograft models and patient samples. EGFR
inhibitors are predicted to disrupt signaling complexes on the EGFR
protein. The inventor will use primary lung cancer xenograft
systems and samples from patients before and after EGFR inhibitor
therapy to measure differences in EGFR protein-protein
interactions.
[0197] Aim 4: Determine if EGFR protein-protein interactions are
associated with responses to EGFR tyrosine kinase inhibitors in
human lung cancer samples. The inventor hypothesizes that
quantitative measurements of EGFR protein-protein interactions
could be used as biomarkers to predict activity of EGFR kinase
inhibitors in patients with advanced lung cancer. The inventor has
developed PLA assays for EGFR protein-protein interactions in lung
cancer samples and related these measurements to response to EGFR
inhibitors in a group of patients with advanced lung cancer.
Aim 1: Establish Proximity Ligation Assays that Measure EGFR
Protein-Protein Interactions.
[0198] The mass spectrometry data provides a set of proteins
physically interacting with one another that can be translated into
assays in tumor material. The initial set of proteins consisting of
EGFR, Src, ErbB3, Mig6, Grb2, Sts1, and p85 is based on the MS data
and the literature. Aside from EGFR, ErbB3 and p85 were selected as
they are important components of mutant EGFR signaling that lead to
activation of Akt signaling and lung cancer survival (31, 32). Grb2
was chosen based on its role in activating Ras signaling downstream
of EGFR (33, 34). Mig6 and Sts1 were selected based on their
ability to negatively regulate EGFR signaling (35-38). Src is
intimately involved in EGFR signaling and important in EGFR driven
lung cancer (39-41). The inventor will measure the following six
interactions: EGFR:ErbB3, EGFR:Grb2, EGFR:Mig6, EGFR:Sts1, EGFR:Src
and ErbB3:p85. Guidelines for antibody validation described by
David Rimm (42) and summarized below will be followed. Protein
extracts from a panel of lung cancer cell lines with and without
EGFR mutations will be used to test for antibody specificity using
western blots. A panel of cells is examined to avoid reaching
conclusions of specificity based on a single cell line. Antibodies
that appear specific (dominant single band, few other bands outside
MW range of protein) will be selected for further development.
[0199] Immunofluorescence (IF) conditions can be optimized for each
antibody, including a series of antibody dilutions, different sets
of temperatures and incubation times, and different concentrations
of secondary antibody conjugated with fluorescence dye. Patterns of
IF staining will be examined using fluorescence and confocal
microscopy using PC9 cells grown on a 8 or 16 well chamber slides.
The antibodies that pass the test in this run of validation have to
reach three criteria at the same time: 1) the location of cell
compartment must be consistent with those that previous reported
(i.e., membrane/cytoplasm/nuclear); 2) antibodies must be sensitive
enough to give enough signal intensity above background; 3) Every
pair of antibodies must pass the double staining test under the
same conditions which is fundamental to the PLA assay. Two
antibodies from different species work must well under identical
conditions with balanced intensities thus requiring us to screen
different pairs of antibodies based on the information from
commercial sources and the IF results described above.
[0200] Validated antibodies will be used for PLA. An initial
experiment was performed (FIGS. 3A-F). PC9 lung cancer cells
harboring an activating EGFR mutation were evaluated for EGFR:Grb2
binding based on the MS data. Antibodies were selected and
validated with both western blotting and IF as described above. PLA
was then tested to measure these interactions in untreated cells
and in cells treated with EGFR TKI erlotinib. Additional controls
were added such as leaving out either primary antibody or leaving
out secondary antibodies. Strong foci were identified indicative of
EGFR:Grb2 interactions that are significantly reduced by erlotinib
and completely absent in the negative controls. To assess
non-specific noise/background in the PLA assays, at least four
negative controls will be used in parallel: omit one of the two
antibodies in each well, and omit one of two secondary antibodies
conjugated with PLUS or MINUS DNA fragments in each well. A
positive control is also used in each individual test. Results
above used a Leica TCS SP5 AOBS laser scanning confocal microscope.
Analysis of labeled foci in the maximum projection images was
performed using Image Pro Plus version 6.2 (Media Cybernetics,
Inc., Silver Springs, Md.). Regions of interest were derived
manually and by image analysis using a click and grow methodology
to define cell borders and segment individual cells. Histogram
threshholding for the biomarker targeted channels were used to
identify the number of foci and determine the mean intensity of the
foci for each individual cell. Foci are defined as any Alexa 555
labeled object within the cell that has an area of at least 5
pixels over threshold. Other approaches will be considered. PLA
assay creates a perfect fluorescence signal allowing conventional
IF to be adapted to the mature AQUA system. The data collection
with AQUA includes scanning fluorescence signal in the tissue
section with AQUAsition, and transferring image signals into
digital numbers with AQUAanlysis software, which is the newest
version package, AQUA 2.2. Definiens.RTM. Developer v1.2
(Definiens, Munich, Germany) software suite can be used to
automatically find individual cells and analyze them. A plasma
membrane or whole cytoplasm stain may need to be added to
accomplish this task. This will allow for batch processing and
increase image analysis throughput significantly, while retaining
quality control standards necessary for this project.
Aim 2: Characterize EGFR Protein Interactions in Cell and Tumor
Models with Known EGFR Mutation status.
[0201] The inventor proposes that cells and tumors with activating
EGFR mutations will demonstrate constitutive complexes with EGFR
proteins. PLA will be tested to recognize increased protein-protein
interactions of EGFR and interacting proteins in a panel of lung
cancer cell lines with known EGFR mutation status. This will
include cell with activating EGFR mutations (PC9, HCC827, H4006,
H1975, H1650, and H820) and wildtype EGFR (H23, A549, H1299, H460,
etc) from the Lung SPORE cell line bank (total of nearly 50 NSCLC
cell lines). It is expected to find higher degrees of PLA signals
in cells with activating EGFR mutations compared to cells without
EGFR mutations as these mutant EGFR proteins are constitutively
activated and poorly internalized. Also examined will be EGFR PPI
PLA signals in lung cancer cells that have wildtype EGFR but
nonetheless have some degree of sensitivity to EGFR TKI. This
includes H322, H358, H292, and H1648 cells. Intermediate
measurements of EGFR PPI using PLA are expected here. This would
reflect engagement of the EGFR protein with downstream signaling
complexes that occur through mechanisms such as EGFR gene
amplification and/or EGFR ligand stimulation.
[0202] The initial studies in human samples will measure
interactions in patient tumors with and without EGFR mutation tumor
sections. For these pilot studies, 20 cases with known EGFR
mutation and 20 with wildtype EGFR will be chosen. It is expected
to find higher PLA signals in the tumors with mutations and this
will provide experience and confidence with human samples. To guide
assay refinement in FFPE specimens, control tissue sections from
PC9 cell xenografts will be included. Tumors from untreated mice
and mice exposed to erlotinib will be collected--the erlotinib
treated mice will have EGFR inhibition and loss of protein-protein
interactions. Thus, they serve as a control where it is expected to
see reduced PLA intensity. This will offer both positive and
negative/down-regulated control for the downstream patient tissue
samples, and also serve as a materials to optimize conditions for
different antibodies for immunohistochemistry (IHC). This is done
even if all the antibodies worked well by IF in cell lines.
Aim 3: Characterize Changes in EGFR Protein-Protein Interactions in
Response to EGFR Tyrosine Kinase Inhibitors in Primary Lung Cancer
Xenograft Models and Patient Samples.
[0203] EGFR inhibitors are predicted to disrupt signaling complexes
on the EGFR protein. Protein interactions for some of these
proteins can be inhibited by EGFR tyrosine kinase inhibitors
(erlotinib). For example, the mass spectrometry data found that
EGFR no longer bound Grb2, Mig6 or Sts1 with treatment of cells
with erlotinib (data not shown). Primary lung cancer xenograft
systems and samples from patients before and after EGFR inhibitor
therapy will be used to measure differences in EGFR protein-protein
interactions. This could allow the development of PLA based assays
as pharmacodynamic markers of drug activity. First, primary lung
cancer explant models will be used. A series of primary human NSCLC
xenograft models from fresh patient tumor samples has been
developed. Detailed morphological and molecular studies
demonstrated that tumors that grew in the xenograft lines closely
resemble the primary tumors, thus, the serially passaged xenografts
provide a more clinically relevant model than established cell
lines. Of the 11 models, 5 have KRAS mutations and one has a drug
sensitive EGFR mutation. Tumors from each mouse will have a core
biopsy before and after 7 days of erlotinib (100 mg/kg/day oral
gavage). Tumors will be formalin fixed and sectioned for PLA
assays. Here it is expected to find reductions in proteins in
complex with EGFR as erlotinib would reduce the levels of tyrosine
phosphorylated EGFR and disrupt binding of proteins such as Grb2 or
Mig6. Second, patient samples treated with EGFR TKI will be used.
The inventor just reported a pilot study of gefitinib in early
stage NSCLC where patients received 4 weeks of gefitinib prior to
having surgical resection of lung cancer (43). This study procured
pre-treatment biopsies (FFPE) and post-treatment biopsies (FFPE
sample of surgical specimen) for each of 23 patients on the study.
Changes in EGFR PPI will be determined using PLA on these tumor
samples. Measurements of EGFR PPI using PLA will be analyzed from
the pre-treatment sample and again on the post-treatment sample.
Here, it is expected that gefitinib will reduce interactions with
EGFR.
Aim 4: Determine if EGFR Protein-Protein Interactions are
Associated with Responses to EGFR Tyrosine Kinase Inhibitors in
Human Lung Cancer Samples.
[0204] The inventor proposes that quantitative measurements of EGFR
protein-protein interactions can be used as biomarkers to predict
activity of EGFR kinase inhibitors in patients with advanced lung
cancer. PLA assays for EGFR protein-protein interactions in lung
cancer samples will be developed and these measurements will be
related to response to EGFR inhibitors in a group of patients with
advanced lung cancer. The Moffitt Lung Cancer Program and SPORE has
outstanding access to tumor samples for these studies. The inventor
has identified 822 patients treated with EGFR TKI at Moffitt in the
last 15 years. Of these patients, 193 have tissue available at
Moffitt. Tissue microarrays will be created from these samples and
used for PLA based assays. The inventor's laboratory has experience
with constructing tissue microarrays used for multiple studies of
biomarkers related to patient outcomes (44-47). The inventor will
correlate PLA biomarkers with response to EGFR TKI. Tumors from
patients who received EGFR TKI (erlotinib or gefitinib) will be
tested for PLA biomarkers and intensity related to (i) response
measured by RECIST and (ii) disease-free survival and (iii) overall
survival. It is expected that patients with higher levels of PLA
biomarkers to have better responses and better survival with EGFR
TKI. To avoid the problem of identifying a prognostic biomarker, a
cohort of patients who received cytotoxic chemotherapy as their
treatment will also be examined. Here it is expected to find no
difference if (i) PLA biomarkers are predictive of outcome for EGFR
TKI and (ii) not prognostic in advanced lung cancer. The SPORE is
currently producing a second TMA for patients with advanced stage
IV non-small cell lung cancer who received chemotherapy at Moffitt.
This includes a cohort of nearly 200 cases of patients that had a
prior surgical procedure and thus have tissue blocks with adequate
tissue to produce tissue arrays. One of the SPORE pathologists is
currently building these TMA and clinical data on these patients is
being annotated through the SPORE infrastructure. Data elements
include age, sex, race, histology, clinical and pathological stage
at presentation, sites of metastatic disease, response to therapy
(by RECIST) and overall survival times.
[0205] Statistical Considerations and Approaches: Proximity
ligation assay (PLA) will be used to quantify EGFR protein-protein
interactions. PLA's measurement consists of number of foci and
signal intensities for each focus. Histograms and boxplots will be
used to visualize and examine the sampling distributions of number
of foci, total intensities, and mean intensity per focus. Normality
of the data will be examined using the Anderson-Darling test.
Proper transformation such as log or square root will be explored
before applying parametric tests to the data. The goal for specific
Aim 2 is to correlate each EGFR protein interaction of interests,
quantified using PLA, to the EGFR mutation status of cell lines.
After proper transformation, two-sample t-test will be performed to
examine whether the signal intensities differ between cell lines
with and without EGFR mutations. When the assumption of normality
is violated, Mann-Whitney U tests will be applied. Limited
attention has been given to the potential of using PLA measurements
as biomarkers in the past, and the utility of using the number of
foci as potential biomarkers is being explored. After examining the
sampling distribution, if appropriate, it will be investigated if
the number of foci differs between cell lines with and without EGFR
mutations using Poisson regression. Over-dispersion will be
examined. With a maximum of 6 proposed interactions in this work,
final number of tests (i.e., total number of interactions) will be
determined by assay development for Aim 1. The same analysis
procedure will be carried out for the data collected from clinical
samples. The goal for specific Aim 3 is to assess if there is a
difference for each protein interaction, quantified by PLA, before
and after EGFR TKI (erloitnib) treatment. The Pre- and
post-treatment samples will be taken from the same xenograph mouse.
Paired t-tests or Wilcoxon signed-rank tests will be performed to
examine the difference of the signal intensities between pre- and
post-treatment for each EGFR protein interaction. The same analysis
procedure will be performed for the clinical samples. The goal for
specific Aim 4 is to identify if each of the EGFR protein-protein
interactions is a predictive factor to EGFR tyrosine kinase
inhibitors (TKI) but not a general prognostic factor (following the
definitions provided by Clark(48, 49). Briefly, a predictive factor
has a differential benefit from the TKI treatment that depends on
the status of the predictive biomarker while a prognostic factor is
thought of as a measure of the natural history of the disease. In
statistical terms, a predictive factor could be identified when the
interaction term between treatment group and biomarker status in
the statistical model is significant. The EGFR protein-protein
interactions will be quantified for 193 human lung cancer samples
from patients with advanced stage and about same amount of samples
from patients treated with chemotherapy using the tissue microarray
arrays (TMAs). The inventor will first examine if there is any
spatial heterogeneity visually using heat map as well as
analytically using regression models (50, 51). Each EGFR
protein-protein interaction will be classified as high- or
low-expression group by searching the optimal cut point using the
maximal logrank method with adjusted P-values for multiple looks,
the association between the overall survival of patients and high-
and low-expression groups will be assessed using the Kaplan-Meier
curve and log-rank test. Gender, histology, and smoking status will
be included along with the treatment group (EGFR TKI vs.
chemotherapy) and biomarker status (high vs. low) in the
multivariable Cox regression model. The interaction term between
treatment group and biomarker status will be tested to determine if
the EGFR protein-protein interaction is a predictive marker.
Stratified analyses will also be performed and facilitate the
visualization of the differential clinical benefit for the patients
in different biomarker group using Kaplan Meier curve. For each
pair of EGFR protein-protein interaction, one Cox multivariable
regression model will first be performed. The assumption of
proportional hazards will be evaluated by examining residual plots
and by including time-dependent terms in the models. Similar
analyses will be performed for the disease free survival. After
proper transforming the signal intensity for each EGFR
protein-protein interaction, one-way ANOVA will be performed to
examine the intensity difference among different response groups
(CR/PR, SD, and PD). When parametric assumptions are violated,
KruskalWallis test will be performed. The overall goal is to
develop and evaluate the proximity ligation assays for each pair of
protein interactions to examine if they are effective biomarkers.
Multiple hypothesis testing will be addressed using 10% false
discovery rate to declare statistical significance. Limited
preliminary data are currently available, and no statistical power
is calculated for sample size justification. The experiments
proposed in this work will give us better estimate of the effective
size and sample size for future work. To examine if each
protein-protein interaction is a good biomarker to classify disease
control (CR/PR/SD) from disease progression (PD), the inventor will
evaluate the sensitivity and specificity. ROC analysis will be
performed to evaluate the area under the curve (AUC) considering
different thresholds. Combination of several markers may lead to a
more accurate classification, all selected and quantified
biomarkers will be included and examined using several
multivariable models such as logistic regression and methods with
built-in variable selection procedures such as Hybrid huberized
support vector machines (HHSVM) and Bayesian variable selection
approach (52, 53).
[0206] The rich MS data that has been experimentally generated for
protein pairs can be capitalized upon to evaluate and test for
interaction. Numerous papers have been published on PLA (23, 25-27,
54). Small interfering RNA (siRNA) molecules can be used to
eliminate proteins in PLA assays as another method to develop
specific assays (55). The inventor expects that siRNA treated cells
will show reduced or absent signaling from PLA assays demonstrating
specificity of the assay against the interaction studied. The
inventor has substantial experience with AQUA; HistoRx will provide
additional expertise and advice, such as providing lists of
validated antibodies and help with troubleshooting (46, 47,
56-58).
[0207] The SPORE and Moffitt Total Cancer Care initiative have
standard operating protocols to minimize these variables. Spatial
heterogeneity in interactions measured by PLA could be observed
across a tumor. Core to core variation can be examined as it is
typical to have 3 cores per tumor for the tissue microarrays. Whole
sections of tumors can also be examined to determine spatial
variability of PLA signals within the same tumor. The PLA signals
may be relatively weak compared to those in conventional AQUA
targets detected with IF. However, one advantage of the new version
of AQUA is better dynamic range that can distinguish those with
higher and lower in PPI signal detected with PLA even with a lower
cutoff. Finally, most of the interactions being examined are based
on phosphorylation dependent interactions (for example,
phosphotyrosine motifs interacting with Src homology-2 (SH2)
domains). Thus, some signals could be weak if samples are not
prepared well and phosphorylation maintained. The ability exists to
examine other signals that are likely more stable based on the MS
data (FIGS. 1A-1D); this includes Hsp90:EGFR, Hsp90:Cdc37, and
other binary interactions.
[0208] FIGS. 3A-F show results of an EGFR-GRB2 PLA in situ. FIGS.
4A-B show results of EGFR-Grb2 PLA in the PC9 lung cancer cell
line. FIGS. 5A-B show results of EGFR-Grb2 PLA in lung cancer tumor
tissues (in vivo). FIGS. 6A-B show results of ALK-Grb2 PLA in H3122
cells. FIGS. 7 and 8 show, respectively, results of EGFR:Grb2 PLA
in cell lines related to EGFR mutation status, and results of
EGFR:Grb2 in the 21 tissue microarray related to EGFR and KRAS
mutation.
EXAMPLE 2
Duolink to Test Egfr and Grb2 Interaction
Reagent Information:
Antibody:
[0209] rGRb2 (Rabbit polyclone: C23, SC-255 Santa Cruz), [0210]
mEGFR (mouse monoclonal antibody, LS-C88071/21583, LIFESPAN
Biosciences) [0211] Working dilutions: m-EGFR (1:250)+r-GRb2
(1:100) [0212] Each well is one square cm, so at least 44 ul/well
of PLA solution is needed. But 100 ul for antibodies and 250 ul for
washing solutions are applied.
Experimental Procedure:
[0212] [0213] 1. Plate approximately 3.times.10.sup.4 cells in 350
.quadrature.01 media per well of a Lab-Tek II 8-well chamber slide.
Grow cells for 36 hours. [0214] 2. Aspirate media and add 250 .mu.l
4% paraformaldehyde in PBS per well with a subsequent incubation
for 20 minutes at 4.degree.. [0215] 3. Aspirate the
paraformaldehyde, then go to step 4. [0216] 4. 250 .mu.l 0.5%
Triton-X 100 per well from a 10% stock diluted in PBS with a
subsequent incubation for 10 minutes at room temperature with
gently rocking. Aspirate the Triton-X 100, wash each well once with
250 .mu.l PBS, and add 250.beta.1 1.5% BSA in PBS (from 10.times.
stock) per well for blocking with gentle rocking for 30 minutes at
room temperature. [0217] 5. Add the appropriate primary antibodies
[m-EGFR (1:250)+r-GRb2 (1:100)] in PBS and incubate at room
temperature for 2 hours with gentle rocking. [0218] 6. Aspirate the
primary antibody solution and wash each well briefly, 3.times. with
250 .mu.l of 1.times. PBS. [0219] 7. Secondary antibody-PLA probes:
Mix and dilute the two PLA probes 1:5 in PBS (9 .mu.l minus +9
.mu.l plus +27 .mu.l PBS=45/well but 44/well, (otherwise the wells
could not be covered), incubate the slides in a pre-heated humidity
chamber for 1 hour at 37C. [0220] 8. Ligation: Dilute the Ligation
stock 1:5 in MQW while waiting (9 .mu.l minus +36 .mu.l MQW=45).
Aspirate the PLA probes and wash 2.times.2minutes in PBS rocking.
Then, add Ligase into ligation solution 1:40 then add 44
.mu.l/well, immediately incubate for 30 minutes at 37C. [0221] 9.
Amplification-hybridations-fluorecences probes: Dilute the
Amplification stock 1:5 in MQW while waiting (9 .mu.lminus +36
.mu.l MQW=45). Aspirate the Ligation solution and wash 2.times.2
minutes in PBS rocking, then add Polymerase 1:80 into amplification
solution, add polymerase-amplification solution 44 .mu.l/well,
incubate for 100 minutes at 37 degrees C. (avoid light). [0222] 10.
Aspirate the amplification solution wash with 1.times. Wash Buffer
B for 2.times.10 minutes Wash the slide in 0.1.times. Wash Buffer
B. Let the slides dry at RT in the dark. Mount the slides with
Duolink II Mounting Medium. Dry it for overnight (avoid light
throughout the step). [0223] 11. Observe under confocal
microscopy.
EXAMPLE 3
Duolink to Test Egfr and Grb2 Interaction in Formalin-Fixed
Paraffin-Embedded (ffpe) Tissue/Cell Pellets
Reagent Information:
[0223] [0224] Antibodies for FFPE tissues and FFPE cell pellets:
[0225] mEGFR (LIFESPAN Bioscie,LS-C88071/21583), 1:50 [0226] rGrb2
(Santa Cruz polyclone C23, SC-255), 1:50
Experimental Procedure:
[0226] [0227] 1. Cut slides in 3 um using window type transfer
system and adhesive coated slides (Instrumedics). After UV exposure
for one minute, peel the tape in TPC solvent, than hydration of
tissue/cells section. [0228] 2. Put slides in PT4 media and heat
with microwave for ten minutes, and then cool down for thirty
minutes at room temperature. [0229] 3. Add 250 .mu.l 0.5% Triton-X
100 per well from a 10% stock diluted in PBS with a subsequent
incubation for 10 minutes at room temperature with gently rocking.
Aspirate the Triton-X 100, wash each well once with 250 .mu.l PBS,
and add 250 .mu.l 1.5% BSA in PBS (from 10.times. stock) per well
for blocking with gentle rocking for thirty minutes at room
temperature. [0230] 4. Add the appropriate primary antibodies at
the pre-determined optimal concentration (see the layout above) in
PBS and incubate at room temperature for over night at 4C with
gentle rocking. [0231] 5. Aspirate the primary antibody solution
and wash the slides briefly three times with 250 .mu.l in PBS.
[0232] 6. Prepare and incubate secondary antibody-PLA probes: Mix
and dilute the two PLA probes 1:5 in PBS (9 .mu.l minus +9 .mu.l
plus +27 ul PBS=45/well but 44/squar cm to cover the whole
section), add 88u1 Mixed PLA probes/well, incubate the slides in a
pre-heated humidity chamber for one hour at 37 degrees C. [0233] 7.
Ligation: Dilute the Ligation stock 1:5 in MQW while waiting (9 ul
minus +36 .mu.MQW=45 .mu.l). Aspirate the PLA probes and wash two
times, each for 2 minutes in PBS with gently rocking, Then, add
ligase into ligation solution 1:40 then add 44 .mu.l/section,
immediately incubate for thirty minutes at 37 degrees C. [0234] 8.
Prepare and incubate the amplification-hybridization-fluorescence
probes: Dilute the amplification stock 1:5 in MQW while waiting (9
.mu.l minus +36 MQW=45)., Aspirate the Ligation solution and wash
two minutes in PBS rocking two times, then add polymerase 1:80 into
amplification solution, add polymerase-amplification solution 44
ul/well, incubate for 100 minutes at 37 degrees C. (avoid light).
[0235] 9. Aspirate the Amplification solution, wash with 1.times.
Wash Buffer B for 10 minutes for 2 times. Pip the slides in
0.1.times. Wash Buffer B (Duolink). Then WGA (this step can be
omitted) for five minutes (avoid light). [0236] 10. Wash with PBS
five minutes for three times. Then let the slides dry at room
temperature in a dark chamber for twenty minutes. Mount the dried
slides with Mounting Medium. Dry the slides for overnight before
observation with confocal microscopy.
EXAMPLE 4
Protein-Protein Interactions for all Human Tyrosine Kinases
[0237] Bioinformatic tools were used to identify all reported
protein-protein interactions for all human tyrosine kinases (2,848
interactions). [0238] 1) 90 of TK (tyrosine kinase) protein were
queried from Kinase.com and verified and annotated with gene
symbol. Kinase.com is produced by at the Salk Institute and has the
full complement of protein kinases in any sequenced genome,
including extensive KinBase database, papers and supporting
material from Sugen and the Salk Institute. [0239] 2) The list of
the TK gene symbol was queried with MiMI (Michigan Molecular
Interactions) plug from Cytoscape, an open-source software platform
for visualizing complex networks and integrating these with any
type of attribute data. The network data were save as an Excel
file. The NCIBI MiMI is part of the NIH's National Center for
Integrative Biomedical Informatics (NCIBI), which provides access
to the knowledge and data merged and integrated from numerous
protein interactions databases. [0240] 3) 90 TK proteins have been
reported for interaction with 1,104 proteins, and 2,848
interactions have been reported. The interactions were verified via
the following databases: BIND, CCSB, DIP, GRID, HPRD, IntAct, MDC,
MINT, reactome and PubMed. The interaction network was rebuilt with
Cytoscape.
[0241] Some of the resulting data is presented in Tables 3-5.
Tables 3 and 4 list ninety human tyrosine kinases, with aliases and
accession numbers. Table 5 lists human tyrosine kinase interactions
(node 1=tyrosine kinase; node 2=interacting protein).
TABLE-US-00003 TABLE 3 Human Tyrosine Kinases (with aliases) Gene
.diamond-solid. Species.uparw. Classification.diamond-solid. Other
Names ABL1 Human TK:Abl: c-ab1, c-ABL, JTK7, p150, ABL1, ABL, v-abl
ABL2 Human TK:Abl: ABLL, ARG, ABL2, ABLL ACK Human TK:Ack: ACK1,
TNK2, ACK, ACK1, FLJ44758, FLJ45547, p21cdc42Hs ALK Human TK:ALK:
ALK, CD246, TFG/ALK AXL Human TK:Axl: UFO, Ark, AXL, UFO BLK Human
TK:Src:SrcB MGC10442, BLK, MGC10442 BMX Human TK:Tec: PSCTK3,
PSCTK2, ETK, BMX, PSCTK3 BRK Human TK:Src:SRM PTK6, PTK6, BRK,
FLJ42088 BTK Human TK:Tec: XLA, PSCTK1, IMD1, BPK, ATK, AGMX1, AT,
BTK, MGC126261, MGC126262, XLA CCK4 Human TK:CCK4: PTK7, PTK7, CCK4
CSK Human TK:Csk: CYL, CSK, MGC117393 CTK Human TK:Csk: MGC2101,
MGC1708, Lsk, HYL, HHYLTK, DKFZp434N1212, CHK, MATK, CTK, HYLTK,
MGC2101 DDR1 Human TK:DDR: trkE, TRKE, RTK6, PTK3A, PTK3, NTRK4,
MCK10, EDDR1, DDR, NEP, CAK, DDR1, CD167 DDR2 Human TK:DDR: TYRO10,
NTRKR3, TKT, DDR2, MIG20a, TYRO10 EGFR Human TK:EGFR: ERBB1, ERBB,
EGFRvIII, EGFR, ERBB1, mENA EphA1 Human TK:Eph: EPHT1, EPHT, EPH,
EPHA1, EPHT1, MGC163163 EphA10 Human TK:Eph: EPHA10, FLJ16103,
FLJ33655, MGC43817 EphA2 Human TK:Eph: ECK, EPHA2, ECK EphA3 Human
TK:Eph: HEK4, HEK, ETK1, TYRO4, ETK, EPHA3, HEK4 EphA4 Human
TK:Eph: TYRO1, HEK8, EPHA4, SEK, TYRO1 EphA5 Human TK:Eph:
Hs.31092, Hs.194771, HEK7, EPHA5, CEK7, EHK1, TYRO4 EphA6 Human
TK:Eph: EPHA6, DKFZp434C1418, EPA6, FLJ35246, PRO57066 EphA7 Human
TK:Eph: HEK11, EPHA7, EHK3, HEK11 EphA8 Human TK:Eph: KIAA1459,
HEK3, EEK, EPHA8, KIAA1459 EphB1 Human TK:Eph: Hek6, HEK6, EPHT2,
ELK, NET, EPHB1, FLJ37986 EphB2 Human TK:Eph: Tyro5, Hek5, HEK5,
EPHT3, DRT, ERK, EPHB2, CAPB, MGC87492, PCBC, Tyro5 EphB3 Human
TK:Eph: TYRO6, HEK2, ETK2, EPHB3, TYRO6 EphB4 Human TK:Eph: TYRO11,
MYK1, HTK, EPHB4, TYRO11 EphB6 Human TK:Eph: HEP, EPHB6, HEP,
MGC129910, MGC129911 ErbB2 Human TK:EGFR: c-erbB2, c-erbB-2, TKR1,
NGL, Hs.323910, Hs.103992, HER2, HER-2, NEU, ERBB2, HER-2/neu,
c-erb B2 ErbB3 Human TK:EGFR: Hs.199067, Hs.167387, Hs.167386,
HER3, ERBB3, ErbB-3, MDA-BF-1, MGC88033, c-erbB-3, c-erbB3,
erbB3-S, p180-ErbB3, p45-sErbB3, p85-sErbB3 ErbB4 Human TK:EGFR:
HER4, ERBB4, HER4, MGC138404, p180erbB4 FAK Human TK:FAK: pp125FAK,
FAKpp125, FAK1, FADK, PTK2, FAK, pp125FAK FER Human TK:Fer: TYK3,
NCP94, FER, TYK3 FES Human TK:Fer: FPS, FES, FPS FGFR1 Human
TK:FGFR: LOC51033, N-SAM, FLT2, FLJ14326, CEK, C-FGR, BFGFR, H5,
H4, H3, H2, FLG, FGFR1, CD331, FGFBR, HBGFR, KAL2, N-SAM FGFR2
Human TK:FGFR: TK25, TK14, KGFR, K-SAM, JWS, ECT1, CFD1, CEK3,
BFR-1, BEK, FGFR2, CD332, TK25 FGFR3 Human TK:FGFR: FGFR3, ACH,
CD333, CEK2, HSFGFR3EX, JTK4 FGFR4 Human TK:FGFR: TKF, JTK2, FGFR4,
CD334, MGC20292, TKF FGR Human TK:Src:SrcA SRC2, FGR, FLJ43153,
MGC75096, SRC2, c-fgr, c-src2, p55c-fgr, p58c-fgr FLT1 Human
TK:VEGFR: VEGFR1, VEGFR-1, FLT-1, FLT, FLT1, VEGFR1 FLT3 Human
TK:PDGFR: FLK2, CD135, STK1, FLT3, FLK2 FLT4 Human TK:VEGFR:
VEGFR3, PCL, FLT4, FLT41, VEGFR3 FMS Human TK:PDGFR: CSF-1R, CD115,
CSF1R, C-FMS, CSFR, FIM2, FMS FRK Human TK:Src:Frk RAK, GTK, FRK,
PTK5, RAK FYN Human TK:Src:SrcA MGC45350, SYN, SLK, FYN, MGC45350
HCK Human TK:Src:SrcB p59hck, p56hck, JTK9, HCK IGF1R Human
TK:InsR: JTK13, IGF1R, CD221, IGFIR, JTK13, MGC142170, MGC142172,
MGC18216 INSR Human TK:InsR: INSR, CD220, HHF5 IRR Human TK:InsR:
INSRR, INSRR, IRR ITK Human TK:Tec: LYK, PSCTK2, EMT, ITK, LYK,
MGC126257, MGC126258 JAK1 Human TK:Jak: JAK1A, JAK1, JAK1A, JAK1B
JAK2 Human TK:Jak: JAK2 JAK3 Human TK:Jak: LJAK, L-JAK, JAKL, JAK3,
JAK-3, JAK3_HUMAN, LJAK KDR Human TK:VEGFR: VEGFR2, VEGFR-2, VEGFR,
Hs.KDR, Hs.12337, FLK1, FLK-1, KDR, CD309, VEGFR2 KIT Human
TK:PDGFR: SCFR, CD117, PBT, KIT, C-Kit, SCFR LCK Human TK:Src:SrcB
p56lck, LCK, YT16, p56lck, pp58lck LMR1 Human TK:Lmr: KIAA0641,
AATYK, AATK, KIAA0641, LMR1, LMTK1 LMR2 Human TK:Lmr: KIAA1079,
LMTK2, AATYK2, BREK, KIAA1079, KPI- 2, KPI2, LMR2, cprk LMR3 Human
TK:Lmr: LMTK3, KIAA1883, LMR3, TYKLM3 LTK Human TK:ALK: TYK1, LTK,
TYK1 LYN Human TK:Src:SrcB JTK8, LYN, FLJ26625, JTK8 MER Human
TK:Axl: mer, c-mer, C-MER, MERTK, MER, MGC133349 MET Human TK:Met:
HGFR, C-MET, RCCP2, MET, HGFR MUSK Human TK:Musk: MUSK, MGC126323,
MGC126324 PDGFRa Human TK:PDGFR: PDGFR2, CD140A, PDGFRA, MGC74795,
PDGFR2, Rhe-PDGFRA PDGFRb Human TK:PDGFR: PDGFR1, PDGFR,
PDGF-R-beta, JTK12, CD140B, PDGFRB, PDGFR1 PYK2 Human TK:FAK:
PTK2B, PKB, CADTK, CAKB, CAK_beta, FAK2, PTK, RAFTK, FADK2, PYK2,
RAFTK RET Human TK:Ret: PTC, CDHF12, HSCR1, Hs.168114, Hs.RET,
MEN2A, MEN2B, MTC1, RET51, RET, RET-ELE1, RET51 RON Human TK:Met:
MST1R, CDw136, CDw136, PTK8, RON ROR1 Human TK:Ror: NTRKR1,
dJ537F10.1, ROR1, MGC99659, dJ537F10.1 ROR2 Human TK:Ror: BDB,
BDB1, NTRKR2, ROR2, MGC163394, NTRKR2 ROS Human TK:Sev: ROS1, MCF3,
MCF3, ROS RYK Human TK:Ryk: D3S3195, RYK1, RYK, JTK5, JTK5A, RYK1
SRC Human TK:Src:SrcA SRC1, ASV, SRC, ASV, c-SRC, p60-Src SRM Human
TK:Src:SRM SRMS, C20orf148, SRM, dJ697K14.1 SuRTK106 Human
TK:TK-Unique: DKFZp761P1010, DKFZP761P1010, STYK1, NOK, SuRTK106
SYK Human TK:Syk: SYK TEC Human TK:Tec: PSCTK4, TEC, MGC126760,
MGC126762, PSCTK4 TIE1 Human TK:Tie: TIE, JTK14, TIE1, JTK14 TIE2
Human TK:Tie: VMCM1, TEK, HPK-6, TIE-2, VMCM, CD202B, TIE2, VMCM1
TNK1 Human TK:Ack: TNK1, MGC46193 TRKA Human TK:Trk: NTRK1, TRK,
MTC, TRK-A, DKFZp781I14186, TRK1, TRKA, p140-TrkA TRKB Human
TK:Trk: NTRK2, NTRK2, GP145-TrkB, TRKB TRKC Human TK:Trk: NTRK3,
NTRK3, TRKC, gp145(trkC) TXK Human TK:Tec: BTKL, PSCTK5, RLK, TKL,
TXK, MGC22473, PTK4, TKL TYK2 Human TK:Jak: JTK1, TYK2, JTK1 TYRO3
Human TK:Axl: DTK, RSE, SKY, TIF, TYRO3, BYK, Brt, Tif YES Human
TK:Src:SrcA YES1, C-YES, P61-YES, c-yes, HsT441, Yes ZAP70 Human
TK:Syk: STD, SRK, ZAP-70, ZAP70, TZK, ZAP-70
TABLE-US-00004 TABLE 4 Human Tyrosine Kinases (with accession
numbers) Gene_Symbol Probeset Accession Gene_ID ABL1 202123_s_at
NM_005157 NM_007313 25 ABL2 231907_at 206411_s_at 226893_at
NM_005158 NM_001168237 27 NM_001168239 NM_007314 NM_001136000
NM_001168236 NM_001168238 TNK2 203839_s_at 216439_at AK225786
NM_001010938 NM_005781 10188 1555557_a_at 228279_s_at AB209338
203838_s_at ALK 208211_s_at 208212_s_at NM_004304 238 AXL
202685_s_at 202686_s_at NM_001699 NM_021913 558 BLK 244394_at
236820_at 206255_at NM_001715 NG_023543 BC038555 640 210934_at BMX
206464_at 242967_at NM_203281 NG_013227 NM_001721 660 PTK6
1553114_a_at NM_005975 5753 BTK 205504_at NM_000061 695 PTK7
1555324_at 207011_s_at BC046109 NM_002821 NM_152881 5754 NM_152880
NM_152882 CSK 202329_at NM_001127190 NM_004383 1445 MATK
206267_s_at NM_002378 NM_139354 NM_139355 4145 DDR1 208779_x_at
207169_x_at NM_001202523 NM_001202521 780 210749_x_at 1007_s_at
NM_013993 NM_001202522 NM_001954 NM_013994 DDR2 227561_at 205168_at
NM_001014796 NM_006182 AY423733 4921 EGFR 211550_at 210984_x_at
211551_at NM_201284 NM_201282 NM_201283 1956 211607_x_at
201984_s_at NM_005228 K03193 201983_s_at 1565483_at 1565484_x_at
EPHA1 215804_at 205977_s_at NM_005232 EU826604 2041 EPHA10
1553371_at 236073_at NM_001099439 NM_173641 284656 EPHA2 203499_at
NM_004431 1969 EPHA3 206071_s_at 206070_s_at NM_182644 NM_005233
2042 211164_at EPHA4 227449_at 228948_at 229374_at NM_004438 2043
206114_at EPHA5 215664_s_at 216837_at 237939_at L36644 NM_004439
NM_182472 2044 EPHA6 233184_at 1561396_at NM_173655 NM_001080448
285220 EPHA7 1554629_at 229288_at 238533_at NM_004440 BC027940 2045
206852_at EPHA8 231796_at 1554069_at NM_020526 NM_001006943 2046
EPHB1 230425_at 211898_s_at NM_004441 2047 210753_s_at EPHB2
209588_at 209589_s_at NM_017449 NM_004442 2048 211165_x_at
210651_s_at EPHB3 204600_at 1438_at NM_004443 2049 EPHB4 202894_at
216680_s_at NM_004444 2050 EPHB6 204718_at NM_004445 2051 ERBB2
210930_s_at 216836_s_at NM_001005862 NM_004448 2064 ERBB3
1563252_at 202454_s_at NM_001982 U88360 2065 226213_at 1563253_s_at
ERBB4 206794_at 241581_at 233494_at NM_001042599 AC108220 NM_005235
2066 214053_at 233498_at AK024204 PTK2 1559529_at 208820_at
NM_153831 NM_005607 AC105009 5747 207821_s_at 241453_at BC043202
NM_001199649 FER 227579_at 206412_at AC116428 NM_005246 2241 FES
205418_at NM_001143783 NM_001143785 2242 NM_002005 NM_001143784
FGFR1 215404_x_at 207822_at 226705_at AK024388 NM_023106 NM_015850
M34187 2260 211535_s_at 210973_s_at NM_023105 NM_001174067
207937_x_at NM_001174066 NM_001174065 NM_023110 NM_001174063
NM_001174064 FGFR2 211399_at 203639_s_at 208225_at NM_001144915
NM_001144918 AB030077 2263 208234_x_at 211398_at NM_001144917
AB030075 M87772 211401_s_at 208228_s_at NM_001144916 NM_001144913
203638_s_at 211400_at NM_022970 NM_001144914 AB030073 NM_001144919
NM_000141 FGFR3 204380_s_at 204379_s_at NM_000142 NM_001163213
NM_022965 2261 FGFR4 1554961_at 211237_s_at NM_002011 NM_022963
AF359241 2264 204579_at 1554962_a_at NM_213647 FGR 208438_s_at
NM_001042729 NM_005248 2268 NM_001042747 FLT1 210287_s_at
226497_s_at NM_001159920 NM_002019 2321 222033_s_at 226498_at FLT3
206674_at NM_004119 2322 FLT4 234379_at 210316_at 229902_at
NM_002020 NM_182925 2324 CSF1R 203104_at NM_005211 1436 FRK
207178_s_at NM_002031 2444 FYN 217697_at 210105_s_at NM_002037
NM_153048 AL109916 2534 216033_s_at NM_153047 HCK 208018_s_at
NM_001172129 NM_001172131 3055 NM_002110 NM_001172130 NM_001172133
NM_001172132 IGF1R 208441_at 243358_at 203627_at AF020763 AC055807
NM_000875 3480 225330_at 238544_at 237377_at 237881_at 203628_at
INSR 227432_s_at 226450_at NM_000208 AB208861 AC010526 3643
213792_s_at 243002_at NM_001079817 226212_s_at 226216_at
207851_s_at INSRR 215776_at NM_014215 3645 ITK 211339_s_at
NM_005546 3702 JAK1 1552611_a_at 239695_at NM_002227 BX648044 3716
1552610_a_at 201648_at JAK2 205842_s_at 205841_at NM_004972
BC043187 3717 1562031_at JAK3 211109_at 211108_s_at 207187_at
NM_000215 U31601 3718 227677_at KDR 203934_at NM_002253 3791 KIT
205051_s_at NM_000222 NM_001093772 3815 LCK 204891_s_at 204890_s_at
NM_001042771 NM_005356 3932 AATK 205986_at XR_115154 NM_001080395
XR_111752 9625 LMTK2 235307_at 206223_at 226375_at NM_014916
NG_013375 22853 LMTK3 1557103_a_at NM_001080434 114783 LTK
207106_s_at 217184_s_at NM_002344 NM_206961 NM_001135685 4058 LYN
202625_at 210754_s_at NM_001111097 NM_002350 4067 202626_s_at MERTK
211913_s_at 206028_s_at NM_006343 10461 MET 213807_x_at 203510_at
NM_001127500 NM_000245 4233 211599_x_at 213816_s_at MUSK 207632_at
207633_s_at NM_001166281 NM_001166280 4593 241122_s_at NM_005592
PDGFRA 211533_at 237696_at 1554828_at L25829 AC098587 M22734
BC015186 5156 203131_at 215305_at NM_006206 PDGFRB 202273_at
NM_002609 5159 PTK2B 203110_at 203111_s_at NM_004103 NM_173175
NM_173174 2185 NM_173176 RET 215771_x_at 211421_s_at NM_020630
NM_020975 5979 205879_x_at MST1R 205455_at NM_002447 4486 ROR1
205805_s_at 211057_at NM_001083592 NM_005012 4919 ROR2 231000_at
205578_at NM_004560 BC051273 4920 ROS1 207569_at NM_002944 6098 RYK
214172_x_at 202853_s_at AC107310 NM_001005861 NM_002958 6259
216976_s_at 238210_at SRC 1565082_x_at 237103_at NG_023033
NM_198291 NM_005417 6714 213324_at 1565080_at 221284_s_at
1558211_s_at SRM 201516_at NM_003132 6723 STYK1 221696_s_at
220030_at NM_018423 55359 SYK 226068_at 209269_s_at BX647192
NM_001135052 NM_001174168 6850 207540_s_at 244023_at NM_001174167
NM_003177 TEC 206301_at NM_003215 7006 TIE1 1560657_at 204468_s_at
NM_005424 AL833389 7075 TEK 217711_at 206702_at NM_000459 7010 TNK1
217149_x_at 205793_x_at NM_003985 8711 NTRK1 208605_s_at
NM_001007792 NM_001012331 4914 NM_002529 NTRK2 214680_at 207152_at
236095_at NM_006180 NM_001018065 4915 221796_at 229463_at 221795_at
NM_001007097 NM_001018064 BX649001 NM_001018066 NTRK3 217033_x_at
217377_x_at NM_002530 NM_001012338 4916 215115_x_at 215025_at
NM_001007156 206462_s_at 1557795_s_at 228849_at TXK 206828_at
NM_003328 7294 TYK2 205546_s_at NM_003331 7297 TYRO3 211432_s_at
211431_s_at NM_006293 X72886 7301 1566934_at YES1 202932_at
202933_s_at NM_005433 7525 ZAP70 1555613_a_at 214032_at NM_207519
NM_001079 7535
TABLE-US-00005 TABLE 5 Human Tyrosine Kinase Interactions (node 1 =
tyrosine kinase; node 2 = interacting protein) Node1 Node2
int_DataBase AATK STK39 (GRID) ABL1 ABI1 (BIND; GRID; HPRD) ABL1
ABI2 (GRID; HPRD; MINT) ABL1 ABL2 (BIND; GRID; HPRD) ABL1 ACTA1
(HPRD) ABL1 ADAM15 (GRID; HPRD) ABL1 APBB1 (HPRD) ABL1 APP (HPRD)
ABL1 ATM (DIP; GRID; HPRD; IntAct) ABL1 ATR (GRID) ABL1 BCAR1
(GRID; HPRD) ABL1 BCL2L1 (MINT) ABL1 BCR (BIND; GRID; HPRD) ABL1
BIN1 (HPRD) ABL1 BRCA1 (GRID; HPRD) ABL1 BTK (HPRD) ABL1 C3 (GRID;
HPRD) ABL1 CABLES1 (HPRD) ABL1 CABLES2 (GRID; HPRD) ABL1 CASP9
(HPRD) ABL1 CAV1 (HPRD) ABL1 CBL (GRID; HPRD; MINT) ABL1 CD19
(HPRD) ABL1 CDC2 (HPRD) ABL1 CDK5 (HPRD) ABL1 CDKN1B (HPRD) ABL1
CREB1 (GRID) ABL1 CRK (BIND; HPRD) ABL1 CRKL (GRID; HPRD) ABL1
CTNND2 (GRID; HPRD) ABL1 DDB1 (HPRD) ABL1 DDB2 (HPRD) ABL1 DOK1
(HPRD) ABL1 DOK3 (HPRD) ABL1 EGFR (MINT) ABL1 EP300 (MINT) ABL1
EPHB2 (GRID; HPRD) ABL1 ERBB2 (MINT) ABL1 ERBB3 (MINT) ABL1 ERBB4
(MINT) ABL1 EVL (GRID; HPRD) ABL1 GPX1 (GRID; HPRD) ABL1 GRB10
(GRID) ABL1 GRB2 (GRID; HPRD) ABL1 GRIN2D (HPRD; MINT) ABL1 HCK
(GRID; HPRD) ABL1 INPPL1 (GRID; HPRD) ABL1 JAK1 (GRID; HPRD) ABL1
JAK2 (HPRD) ABL1 JUN (HPRD) ABL1 KIT (HPRD) ABL1 MAP4K1 (HPRD) ABL1
MAP4K5 (GRID; HPRD) ABL1 MAPT (HPRD) ABL1 MDM2 (GRID; HPRD) ABL1
MUC1 (HPRD; MINT) ABL1 NCF1 (BIND) ABL1 NCK1 (GRID; HPRD) ABL1
NCSTN (GRID; HPRD) ABL1 NEDD9 (GRID; HPRD) ABL1 NTRK1 (GRID; HPRD)
ABL1 PAG1 (GRID; HPRD) ABL1 PAK2 (GRID; HPRD) ABL1 PDE4D (BIND)
ABL1 PIK3R1 (BIND; GRID; HPRD) ABL1 PLCG1 (BIND; HPRD) ABL1 PLSCR1
(HPRD; MINT) ABL1 POLR2A (HPRD) ABL1 PRKCD (MINT) ABL1 PRKD1 (HPRD)
ABL1 PRKDC (GRID; HPRD; MINT) ABL1 PSTPIP1 (GRID; HPRD) ABL1 PTPN12
(HPRD) ABL1 PTPN18 (HPRD) ABL1 PTPN6 (GRID; HPRD) ABL1 PXN (GRID;
HPRD) ABL1 RAD51 (GRID; HPRD; MINT) ABL1 RAD52 (HPRD) ABL1 RAD9A
(GRID; HPRD) ABL1 RAN (GRID; HPRD) ABL1 RASA1 (BIND; HPRD) ABL1 RB1
(BIND; GRID; HPRD; MINT) ABL1 RFX1 (GRID; HPRD) ABL1 RIN1 (BIND;
GRID; HPRD; IntAct; MINT) ABL1 ROBO1 (BIND; HPRD) ABL1 ROS1 (GRID;
HPRD) ABL1 RYBP (GRID) ABL1 SFN (HPRD; IntAct) ABL1 SH3BP1 (GRID)
ABL1 SH3BP2 (HPRD) ABL1 SHD (GRID; HPRD) ABL1 SHE (HPRD) ABL1
SLC9A2 (GRID; HPRD) ABL1 SORBS1 (GRID; HPRD) ABL1 SORBS2 (GRID;
HPRD) ABL1 SOS2 (HPRD) ABL1 SRC (BIND; HPRD) ABL1 ST5 (GRID; HPRD)
ABL1 TERT (HPRD; MINT) ABL1 TP53 (GRID; HPRD; MINT) ABL1 TP73
(GRID; HPRD; MINT) ABL1 TRAF6 (HPRD) ABL1 TUB (GRID; HPRD) ABL1
WASF1 (BIND; HPRD; MINT) ABL1 WASL (HPRD) ABL1 XPO1 (BIND) ABL1
XRCC6 (GRID; HPRD) ABL1 YTHDC1 (GRID; HPRD) ABL1 YWHAB (HPRD) ABL1
YWHAE (HPRD) ABL1 YWHAG (HPRD) ABL1 YWHAH (BIND; HPRD) ABL1 YWHAZ
(HPRD) ABL1 ZAP70 (GRID; HPRD) ABL1 ZDHHC16 (GRID; HPRD) ABL2 ABI2
(BIND) ABL2 BCR (BIND; HPRD) ABL2 CAT (GRID; HPRD) ABL2 CRK (BIND;
GRID; HPRD; IntAct) ABL2 EGFR (MINT) ABL2 EPHB2 (HPRD) ABL2 ERBB2
(MINT) ABL2 ERBB3 (MINT) ABL2 ERBB4 (MINT) ABL2 GPX1 (HPRD) ABL2
HCK (GRID; HPRD) ABL2 HRAS (MINT) ABL2 JAK1 (GRID) ABL2 ONECUT1
(BIND) ABL2 RIN1 (BIND; DIP; IntAct; MINT) ABL2 SIVA1 (HPRD) ABL2
SORBS2 (GRID; HPRD) ALK CENPF (IntAct) ALK EIF4B (IntAct) ALK EPHA1
(IntAct) ALK EPHB3 (IntAct) ALK HSP90AA1 (GRID) ALK HSPD1 (IntAct)
ALK IRS1 (GRID; HPRD; IntAct) ALK JAK3 (GRID; HPRD) ALK MAP3K1
(IntAct) ALK MAP3K3 (IntAct; MINT) ALK MDK (HPRD) ALK NPM1 (HPRD)
ALK PLCG1 (GRID; HPRD) ALK PTN (DIP; GRID; HPRD) ALK PTPRZ1 (HPRD)
ALK RASA1 (GRID) ALK SHC1 (GRID; HPRD) ALK SHC3 (GRID; HPRD) ALK
SOCS5 (IntAct) ALK STAT3 (IntAct) ALK TNFRSF8 (HPRD) ALK ZC3HC1
(HPRD) AXL ADAM10 (HPRD) AXL CBL (HPRD) AXL CSK (BIND) AXL GAS6
(GRID; HPRD; MINT) AXL GRB2 (BIND; CCSB; GRID; HPRD) AXL IL15RA
(HPRD) AXL IL2RG (HPRD) AXL LCK (BIND; CCSB; GRID; HPRD) AXL NCK2
(BIND; HPRD) AXL PIK3R1 (BIND; HPRD) AXL PIK3R2 (BIND; GRID; HPRD)
AXL PIK3R3 (BIND; HPRD) AXL PLCG1 (BIND; HPRD) AXL PTPN11 (HPRD)
AXL RANBP9 (BIND; HPRD) AXL SHC1 (BIND; CCSB; GRID; HPRD) AXL SHC3
(HPRD) AXL SOCS1 (BIND; HPRD) AXL SRC (CCSB; GRID; HPRD) AXL TENC1
(BIND; GRID; HPRD) BLK BCAS2 (GRID; HPRD) BLK BCL2 (GRID; HPRD) BLK
BCR (GRID; HPRD) BLK CBL (GRID; HPRD; MINT) BLK CD79A (GRID; HPRD)
BLK CD79B (GRID; HPRD) BLK CTR9 (GRID; HPRD) BLK EGFR (MINT) BLK
ERBB2 (MINT) BLK FCGR2A (GRID; HPRD) BLK FCGR2B (HPRD) BLK MAX
(BIND) BLK MYC (BIND) BLK PLCG2 (GRID; HPRD) BLK TAF1 (BIND) BLK
UBE3A (GRID; HPRD) BMX BTK (HPRD) BMX CASP3 (CCSB; GRID; HPRD) BMX
CAV1 (CCSB; GRID: HPRD) BMX EGFR (MINT) BMX ERBB2 (MINT) BMX ITK
(HPRD) BMX KDR (HPRD) BMX PAK1 (GRID; HPRD) BMX PIM1 (IntAct) BMX
PTK2 (CCSB; GRID; HPRD) BMX PTPN21 (GRID; HPRD) BMX RAP1A (HPRD)
BMX RUFY1 (BIND; CCSB; GRID; HPRD) BMX RUFY2 (HPRD) BMX SRC (CCSB;
GRID; HPRD) BMX STAT1 (CCSB; GRID; HPRD) BMX STAT3 (CCSB; GRID;
HPRD) BMX STAT5A (CCSB; GRID; HPRD) BMX TEC (HPRD) BMX TNFRSF1B
(HPRD) BMX TP53 (IntAct) BTK ARID3A (GRID; HPRD) BTK BLNK (GRID;
HPRD; MINT) BTK CAV1 (GRID; HPRD) BTK CBL (BIND; GRID; HPRD) BTK
CD19 (HPRD) BTK CMTM3 (HPRD) BTK DAPP1 (HPRD) BTK EWSR1 (GRID;
HPRD) BTK FAS (GRID; HPRD) BTK FCER1G (IntAct) BTK GNA12 (GRID;
HPRD) BTK GNAQ (GRID; HPRD) BTK GNG2 (HPRD) BTK GTF2I (GRID; HPRD;
IntAct) BTK HCK (GRID; HPRD) BTK IBTK (GRID; HPRD) BTK IRAK1 (HPRD)
BTK ITK (HPRD) BTK JAK1 (GRID; HPRD) BTK KHDRBS1 (GRID; HPRD) BTK
KIT (HPRD) BTK LYN (GRID; HPRD) BTK MAPK1 (HPRD) BTK MYD88 (HPRD)
BTK PIK3AP1 (HPRD) BTK PIP4K2A (HPRD) BTK PIP4K2B (HPRD) BTK
PIP4K2C (HPRD) BTK PIP5K1A (HPRD) BTK PIP5K1B (HPRD) BTK PIP5K1C
(HPRD) BTK PLCG1 (HPRD) BTK PLCG2 (HPRD)
BTK PRKCA (HPRD) BTK PRKCB1 (HPRD) BTK PRKCE (HPRD) BTK PRKCQ
(HPRD; IntAct) BTK PRKCZ (HPRD) BTK PRKD1 (BIND; GRID; HPRD) BTK
RELA (HPRD) BTK SH2B2 (HPRD) BTK SH3BP5 (GRID; HPRD; IntAct) BTK
STAT5A (HPRD) BTK SYK (HPRD) BTK TAF1 (BIND) BTK TEC (HPRD) BTK
TIRAP (HPRD) BTK TLR4 (HPRD) BTK TLR6 (HPRD) BTK TLR8 (HPRD) BTK
TLR9 (HPRD) BTK TNFRSF10A (HPRD) BTK VAV1 (GRID; HPRD) BTK WAS
(GRID; HPRD) BTK WASF2 (GRID; HPRD) CSF1R CBL (GRID; HPRD) CSF1R
CSF1 (GRID; HPRD) CSF1R FYN (GRID; HPRD) CSF1R GRAP2 (GRID; HPRD)
CSF1R GRB2 (GRID; HPRD) CSF1R INPP5D (BIND; HPRD) CSF1R INPPL1
(BIND; HPRD) CSF1R LYN (GRID; HPRD) CSF1R PIK3R1 (BIND; GRID; HPRD)
CSF1R PIK3R2 (GRID; HPRD) CSF1R RASA1 (GRID; HPRD) CSF1R RUNX1
(BIND) CSF1R SHC1 (HPRD) CSF1R SOCS1 (BIND; GRID; HPRD) CSF1R SOCS3
(GRID; HPRD) CSF1R SOS1 (HPRD) CSF1R THOC5 (HPRD) CSF1R YES1 (GRID;
HPRD) CSK ADRB2 (HPRD) CSK ARRB1 (GRID; HPRD) CSK CAV1 (GRID; HPRD)
CSK CBL (GRID) CSK CD247 (GRID; HPRD) CSK CD44 (GRID; HPRD) CSK
CDH5 (BIND; HPRD) CSK CREBBP (GRID; HPRD) CSK DAB2 (GRID; HPRD) CSK
DOK1 (HPRD) CSK DOK3 (HPRD) CSK EGFR (BIND; GRID; MINT) CSK ERBB3
(BIND; GRID; MINT) CSK FGR (HPRD) CSK FYN (GRID; HPRD) CSK GJA1
(HPRD; MINT) CSK GSN (HPRD) CSK HCK (HPRD) CSK HNF4A (BIND) CSK
HNRPK (GRID; HPRD) CSK IGF1R (BIND; GRID; HPRD; MINT) CSK INSR
(BIND; GRID; HPRD) CSK LAIR1 (HPRD) CSK LCK (HPRD) CSK LYN (HPRD)
CSK MAPK15 (HPRD) CSK PAG1 (GRID; HPRD) CSK PARD3 (MINT) CSK PECAM1
(GRID; HPRD) CSK PLD2 (GRID; HPRD) CSK PRKACA (HPRD) CSK PTK2
(GRID; HPRD) CSK PTPN12 (GRID; HPRD) CSK PTPN18 (GRID; HPRD) CSK
PTPN22 (HPRD) CSK PXN (GRID; HPRD) CSK RASA1 (GRID; HPRD) CSK RB1
(HPRD) CSK RGS16 (BIND; HPRD) CSK SDC3 (GRID; HPRD) CSK SHC1 (GRID;
HPRD) CSK SIT1 (GRID; HPRD) CSK SRC (GRID; HPRD) CSK YES1 (HPRD)
CSK YTHDC1 (GRID) DDR1 C1orf165 (HPRD; IntAct) DDR1 COL11A1 (GRID;
HPRD) DDR1 COL2A1 (GRID; HPRD) DDR1 COL3A1 (GRID; HPRD) DDR1 COL5A2
(GRID; HPRD) DDR1 FRS2 (GRID; HPRD) DDR1 NCK2 (HPRD; MINT) DDR1
PLCG1 (GRID; HPRD) DDR1 PTPN11 (HPRD; MINT) DDR1 RGS2 (HPRD;
IntAct; MDC; MINT) DDR1 SHC1 (GRID; HPRD) DDR1 SNAPIN (HPRD;
IntAct; MDC; MINT) DDR1 TM4SF1 (HPRD; IntAct; MDC; MINT) DDR1 TTR
(HPRD; IntAct; MDC; MINT) DDR1 WDR57 (HPRD; IntAct; MDC; MINT) DDR2
COL1A1 (GRID; HPRD) DDR2 COL3A1 (GRID; HPRD) DDR2 SHC1 (GRID; HPRD;
MINT) DDR2 SRC (GRID; HPRD; MINT) EGFR ACTA1 (HPRD) EGFR ADAM10
(reactome) EGFR ADAM12 (reactome) EGFR ADAM17 (reactome) EGFR
ADRBK1 (MINT) EGFR AGTR1 (HPRD) EGFR ALCAM (HPRD) EGFR AMH (GRID;
HPRD) EGFR ANKS1A (MINT) EGFR ANXA1 (HPRD; MINT) EGFR APBA3 (MINT)
EGFR APBB1 (MINT) EGFR APBB2 (MINT) EGFR APBB3 (MINT) EGFR APPL1
(MINT) EGFR AR (GRID; HPRD) EGFR AREG (DIP; GRID; HPRD) EGFR ARF4
(GRID; HPRD) EGFR ATP1A1 (HPRD) EGFR ATP1B1 (HPRD) EGFR ATP5C1
(HPRD; IntAct; MDC; MINT) EGFR BTC (DIP; GRID; HPRD) EGFR CALM1
(BIND; HPRD) EGFR CALM2 (HPRD) EGFR CALM3 (HPRD) EGFR CAMK2A (HPRD)
EGFR CAMK2G (HPRD) EGFR CAMLG (GRID; HPRD) EGFR CASP1 (GRID; HPRD)
EGFR CAV1 (GRID; HPRD) EGFR CAV2 (HPRD) EGFR CAV3 (GRID; HPRD) EGFR
CBL (BIND; HPRD; IntAct; MINT) EGFR CBLB (GRID; HPRD) EGFR CBLC
(GRID; HPRD) EGFR CD44 (BIND; GRID; HPRD) EGFR CD59 (IntAct) EGFR
CD82 (GRID; HPRD) EGFR CDC25A (GRID; HPRD) EGFR CDH1 (BIND; GRID;
HPRD) EGFR CEACAM1 (HPRD) EGFR CEBPB (GRID; HPRD) EGFR CISH (MINT)
EGFR CLTA (BIND; HPRD) EGFR CLTCL1 (HPRD) EGFR COX2 (HPRD) EGFR CRK
(BIND; GRID; HPRD; MINT) EGFR CRKL (MINT) EGFR CTNNB1 (GRID; HPRD)
EGFR CTNND1 (GRID; HPRD) EGFR DCN (GRID; HPRD) EGFR DEGS1 (GRID;
HPRD) EGFR DOK2 (GRID; HPRD) EGFR DOK4 (MINT) EGFR DOK5 (MINT) EGFR
DOK6 (MINT) EGFR EGF (BIND; DIP; GRID; HPRD; IntAct; MINT;
reactome) EGFR ELF3 (HPRD) EGFR EPB41 (HPRD) EGFR EPHA2 (HPRD) EGFR
EPPK1 (GRID; HPRD) EGFR EPS15 (GRID; HPRD) EGFR EPS8 (GRID; HPRD)
EGFR ERBB2 (BIND; GRID; HPRD; IntAct; MINT) EGFR ERBB3 (GRID; HPRD;
IntAct) EGFR ERBB4 (HPRD; IntAct) EGFR EREG (GRID; HPRD) EGFR
ERRFI1 (GRID; HPRD) EGFR ESR1 (GRID; HPRD) EGFR EZR (HPRD) EGFR FAS
(HPRD) EGFR FER (GRID; HPRD) EGFR FES (MINT) EGFR FRS2 (HPRD) EGFR
GAB1 (GRID; HPRD) EGFR GAB2 (IntAct) EGFR GAPDH (HPRD) EGFR GNAI2
(GRID; HPRD) EGFR GRB10 (BIND; GRID; HPRD; MINT) EGFR GRB14 (GRID;
HPRD) EGFR GRB2 (BIND; DIP; GRID; HPRD) EGFR GRB7 (GRID; HPRD) EGFR
HBEGF (GRID; HPRD) EGFR HGS (HPRD) EGFR HIST3H3 (HPRD) EGFR HOXC10
(HPRD) EGFR HSP90AA1 (BIND) EGFR HTT (GRID; HPRD) EGFR ICAM1 (HPRD)
EGFR INPPL1 (GRID; HPRD) EGFR IRS1 (MINT) EGFR IRS4 (MINT) EGFR
ITGA5 (GRID; HPRD) EGFR JAK2 (HPRD; MINT) EGFR JUP (GRID) EGFR
KRT17 (GRID; HPRD) EGFR KRT18 (GRID; HPRD) EGFR KRT7 (GRID; HPRD)
EGFR KRT8 (GRID; HPRD) EGFR LRSAM1 (GRID) EGFR LYN (HPRD) EGFR
MAP2K1 (HPRD) EGFR MAP3K14 (GRID; HPRD) EGFR MAP4K1 (GRID; HPRD)
EGFR MAPK1 (HPRD) EGFR MAPK8IP1 (MINT) EGFR MAPK8IP2 (MINT) EGFR
MET (HPRD) EGFR MIST (MINT) EGFR MUC1 (BIND; GRID; HPRD; MINT) EGFR
NCK1 (GRID; HPRD; MINT) EGFR NCK2 (GRID; HPRD) EGFR NRG1 (GRID)
EGFR NUMB (MINT) EGFR NUMBL (MINT) EGFR PAK1 (BIND; HPRD) EGFR
PDGFRB (GRID; HPRD) EGFR PIK3C2A (IntAct) EGFR PIK3C2B (GRID; HPRD;
IntAct) EGFR PIK3R1 (BIND; HPRD; MINT) EGFR PIK3R2 (BIND; HPRD;
MINT) EGFR PITPNA (GRID; HPRD) EGFR PKIA (HPRD) EGFR PLCG1 (BIND;
GRID; HPRD; MINT) EGFR PLCG2 (MINT) EGFR PLD1 (HPRD) EGFR PLD2
(BIND; GRID; HPRD) EGFR PLEC1 (GRID; HPRD) EGFR PLSCR1 (GRID; HPRD;
MINT) EGFR PRKACA (GRID; HPRD) EGFR PRKAR1A (GRID; HPRD) EGFR PRKCA
(GRID; HPRD) EGFR PRKCD (HPRD) EGFR PRKD1 (HPRD) EGFR PTK2 (BIND;
GRID; HPRD) EGFR PTK2B (GRID; HPRD) EGFR PTK6 (GRID; HPRD; MINT)
EGFR PTPN1 (GRID; HPRD) EGFR PTPN11 (BIND; GRID; HPRD; MINT) EGFR
PTPN2 (HPRD) EGFR PTPN6 (GRID; HPRD) EGFR PTPRJ (GRID; HPRD) EGFR
PTPRS (HPRD) EGFR RASA1 (BIND; GRID; HPRD; MINT) EGFR RGS16 (GRID;
HPRD) EGFR RIN2 (MINT) EGFR RIPK1 (GRID; HPRD)
EGFR S100A7 (HPRD) EGFR S100A9 (HPRD) EGFR SCAMP1 (BIND; HPRD) EGFR
SCAMP3 (BIND; HPRD) EGFR SEC13 (HPRD) EGFR SFN (HPRD) EGFR SGSM2
(HPRD; MDC) EGFR SH2B3 (MINT) EGFR SH2D1A (MINT) EGFR SH2D2A (MINT)
EGFR SH2D3A (GRID; HPRD) EGFR SH3BGRL (GRID; MINT) EGFR SH3BGRL3
(BIND) EGFR SH3GL2 (HPRD) EGFR SH3KBP1 (HPRD) EGFR SHC1 (BIND; DIP;
GRID; HPRD; IntAct; MINT) EGFR SHC2 (MINT) EGFR SHC3 (GRID; HPRD;
MINT) EGFR SLC3A2 (HPRD) EGFR SNRPD2 (GRID; HPRD) EGFR SNX1 (DIP;
GRID; HPRD) EGFR SNX2 (GRID; HPRD) EGFR SNX4 (GRID; HPRD) EGFR SNX6
(GRID; HPRD) EGFR SOCS1 (GRID; HPRD) EGFR SOCS3 (GRID; HPRD) EGFR
SOS1 (GRID; HPRD) EGFR SOS2 (GRID; HPRD) EGFR SRC (GRID; HPRD;
reactome) EGFR STAM2 (HPRD) EGFR STAT1 (GRID; HPRD) EGFR STAT3
(BIND; GRID; HPRD) EGFR STAT5A (GRID; HPRD) EGFR STAT5B (BIND;
GRID; HPRD; MINT) EGFR SYK (MINT) EGFR TGFA (DIP; GRID; HPRD;
IntAct) EGFR TJP1 (GRID; HPRD) EGFR TLN1 (MINT) EGFR TLR2 (MINT)
EGFR TLR4 (MINT) EGFR TNC (GRID; HPRD) EGFR TNK2 (GRID; HPRD) EGFR
TNS4 (MINT) EGFR TUBA4A (HPRD) EGFR UBB (HPRD) EGFR VAV1 (GRID;
HPRD) EGFR VAV2 (BIND; GRID; HPRD) EGFR VAV3 (GRID; HPRD) EGFR XPO6
(HPRD) EGFR XRCC6 (GRID; HPRD) EGFR YWHAZ (BIND; HPRD) EGFR ZAP70
(MINT) EGFR ZNF259 (GRID; HPRD) EPHA1 EFNA1 (BIND; GRID; HPRD)
EPHA1 HNF4A (BIND) EPHA1 ONECUT1 (BIND) EPHA1 SMURF2 (BIND; HPRD)
EPHA2 ACP1 (GRID; HPRD) EPHA2 CBL (HPRD) EPHA2 CDH5 (HPRD) EPHA2
CLDN4 (HPRD) EPHA2 EFNA1 (BIND; DIP; GRID; HPRD) EPHA2 EFNA2 (GRID;
HPRD) EPHA2 EFNA3 (BIND; GRID; HPRD) EPHA2 EFNA4 (BIND; GRID; HPRD)
EPHA2 EFNA5 (BIND; GRID; HPRD) EPHA2 GRB2 (GRID; HPRD) EPHA2 HNF4A
(BIND) EPHA2 PAX3 (BIND) EPHA2 PIK3R1 (GRID) EPHA2 PIK3R2 (HPRD)
EPHA2 PTK2 (GRID; HPRD) EPHA2 PTPN11 (GRID; HPRD) EPHA2 RASA1
(HPRD) EPHA2 SHC1 (GRID; HPRD) EPHA2 SLA (GRID; HPRD) EPHA2 TIAM1
(HPRD) EPHA2 TNFAIP1 (GRID; HPRD) EPHA3 ADAM10 (HPRD) EPHA3 CRK
(GRID; HPRD) EPHA3 E2F4 (BIND) EPHA3 EFNA1 (HPRD) EPHA3 EFNA2
(GRID; HPRD) EPHA3 EFNA5 (GRID; HPRD) EPHA3 EFNB2 (GRID; HPRD)
EPHA3 RUFY1 (GRID; HPRD) EPHA3 RUFY2 (GRID; HPRD) EPHA3 TP53 (GRID)
EPHA4 ARHGEF15 (CCSB; GRID; HPRD) EPHA4 EFNA1 (BIND; CCSB; GRID;
HPRD) EPHA4 EFNA3 (BIND; GRID; HPRD) EPHA4 EFNA4 (BIND; GRID; HPRD)
EPHA4 EFNA5 (BIND; HPRD) EPHA4 EFNB2 (BIND; GRID; HPRD) EPHA4 EFNB3
(GRID; HPRD) EPHA4 FGFR1 (HPRD) EPHA4 FGFR2 (HPRD) EPHA4 FGFR3
(HPRD) EPHA4 FGFR4 (HPRD) EPHA4 FYN (CCSB; GRID; HPRD) EPHA4 NGEF
(CCSB; GRID; HPRD) EPHA4 PAX3 (BIND) EPHA5 EFNA1 (BIND; GRID; HPRD)
EPHA5 EFNA2 (GRID; HPRD) EPHA5 EFNA3 (BIND; GRID; HPRD) EPHA5 EFNA4
(BIND; HPRD) EPHA5 EFNA5 (BIND; GRID; HPRD) EPHA5 STAT3 (GRID)
EPHA6 EFNA1 (BIND; GRID; HPRD) EPHA6 EFNA3 (BIND) EPHA6 EFNA4
(BIND; HPRD) EPHA6 EFNA5 (BIND) EPHA7 EFNA1 (BIND: CCSB; GRID;
HPRD) EPHA7 EFNA3 (BIND; HPRD) EPHA7 EFNA4 (BIND; HPRD) EPHA7 EFNA5
(BIND; HPRD) EPHA7 MLLT4 (BIND; GRID; HPRD; MINT) EPHA7 SDCBP
(BIND) EPHA8 EFNA1 (HPRD) EPHA8 EFNA4 (HPRD) EPHA8 EFNA5 (GRID;
HPRD) EPHA8 FYN (CCSB; GRID; HPRD) EPHA8 PIK3CG (CCSB; GRID; HPRD)
EPHB1 ACP1 (BIND; GRID; HPRD) EPHB1 EFNA1 (HPRD) EPHB1 EFNA5 (GRID;
HPRD) EPHB1 EFNB1 (BIND; GRID; HPRD) EPHB1 EFNB2 (BIND; GRID; HPRD)
EPHB1 EPHB6 (GRID; HPRD) EPHB1 GRB10 (BIND; HPRD; IntAct) EPHB1
GRB2 (BIND; GRID; HPRD) EPHB1 NCK1 (GRID; HPRD) EPHB1 PDGFRB (BIND)
EPHB1 PXN (HPRD) EPHB2 ACP1 (BIND; GRID; HPRD) EPHB2 ADAM17 (GRID)
EPHB2 AQP1 (HPRD) EPHB2 ARHGEF6 (HPRD) EPHB2 BCL2 (GRID) EPHB2
EFNA5 (HPRD) EPHB2 EFNB1 (BIND; GRID; HPRD) EPHB2 EFNB2 (BIND;
GRID; HPRD) EPHB2 ERF (HPRD) EPHB2 FOS (HPRD) EPHB2 GRB2 (HPRD)
EPHB2 GRIN1 (HPRD) EPHB2 GRIN2B (HPRD) EPHB2 ITSN1 (GRID; HPRD)
EPHB2 KALRN (HPRD) EPHB2 MLLT4 (BIND; GRID; HPRD; MINT) EPHB2 NCK1
(GRID; HPRD) EPHB2 PICK1 (HPRD) EPHB2 PTK2 (HPRD) EPHB2 RASA1
(GRID; HPRD) EPHB2 RRAS (HPRD) EPHB2 RYK (GRID; HPRD) EPHB2 SDC2
(HPRD) EPHB2 SDCBP (HPRD) EPHB2 SH2D3C (HPRD) EPHB2 SRC (GRID;
HPRD) EPHB2 SYNJ1 (HPRD) EPHB2 VAV2 (HPRD) EPHB3 CRK (GRID; HPRD)
EPHB3 EFNB3 (GRID; HPRD) EPHB3 FYN (GRID; HPRD) EPHB3 HNRPA3 (BIND)
EPHB3 MLLT4 (BIND; GRID; HPRD; MINT) EPHB3 RASA1 (GRID; HPRD) EPHB3
RYK (GRID; HPRD) EPHB4 EFNB2 (GRID; HPRD) EPHB4 GRIN1 (HPRD) EPHB4
SDC3 (HPRD) EPHB6 CBL (GRID; HPRD) EPHB6 CRK (HPRD) EPHB6 CRKL
(HPRD) EPHB6 EFNB2 (GRID; HPRD) EPHB6 GRB2 (HPRD) EPHB6 HDHD2
(HPRD) EPHB6 HNRPA3 (BIND) EPHB6 MLLT4 (BIND; GRID; HPRD; MINT)
EPHB6 SAT1 (HPRD; IntAct; MDC; MINT) ERBB2 ACPP (HPRD) ERBB2 ANKS1A
(MINT) ERBB2 APBB1 (MINT) ERBB2 APBB3 (MINT) ERBB2 BLNK (MINT)
ERBB2 BTC (GRID; HPRD) ERBB2 CAV1 (GRID; HPRD) ERBB2 CBL (BIND)
ERBB2 CD82 (GRID; HPRD) ERBB2 CDC37 (BIND) ERBB2 CHN1 (MINT) ERBB2
CISH (MINT) ERBB2 CRK (MINT) ERBB2 CRKL (MINT) ERBB2 CTNNB1 (GRID;
HPRD) ERBB2 DAB1 (MINT) ERBB2 DLG4 (GRID; HPRD) ERBB2 DOK1 (MINT)
ERBB2 DOK4 (MINT) ERBB2 DOK6 (MINT) ERBB2 EGF (GRID; HPRD) ERBB2
ERBB2IP (GRID; HPRD) ERBB2 ERBB3 (GRID; HPRD; IntAct) ERBB2 ERBB4
(IntAct) ERBB2 ERRFI1 (BIND; GRID; HPRD) ERBB2 ESR1 (HPRD) ERBB2
FER (MINT) ERBB2 FGR (MINT) ERBB2 GRAP2 (MINT) ERBB2 GRB2 (BIND;
GRID; HPRD) ERBB2 GRB7 (GRID; HPRD; MINT) ERBB2 H3F3A (BIND) ERBB2
HLA-A (GRID; HPRD) ERBB2 HSP90AA1 (BIND; GRID; HPRD) ERBB2 HSP90B1
(GRID; HPRD) ERBB2 HSPA8 (BIND) ERBB2 IL6ST (GRID; HPRD) ERBB2 IRS1
(MINT) ERBB2 IRS4 (MINT) ERBB2 ITGA5 (HPRD) ERBB2 ITGB4 (GRID;
HPRD) ERBB2 ITK (MINT) ERBB2 JAK1 (MINT) ERBB2 JAK2 (GRID; HPRD)
ERBB2 JUP (GRID; HPRD) ERBB2 MAPK8IP1 (MINT) ERBB2 MAPK8IP2 (MINT)
ERBB2 MATK (GRID; HPRD; MINT) ERBB2 MIST (MINT) ERBB2 MMP16 (BIND)
ERBB2 MUC1 (GRID; HPRD) ERBB2 MUC4 (HPRD) ERBB2 NCK2 (MINT) ERBB2
NCOA3 (BIND) ERBB2 NCOR1 (BIND) ERBB2 NF2 (HPRD) ERBB2 NRG1 (DIP;
GRID; MINT) ERBB2 PAK1 (GRID; HPRD) ERBB2 PICK1 (GRID; HPRD) ERBB2
PIK3C2A (IntAct) ERBB2 PIK3C2B (IntAct) ERBB2 PIK3R1 (GRID; HPRD;
MINT) ERBB2 PIK3R2 (GRID; HPRD; MINT) ERBB2 PLCG1 (GRID; HPRD;
MINT) ERBB2 PLCG2 (MINT) ERBB2 PTK2 (GRID; HPRD) ERBB2 PTK2B (GRID;
HPRD) ERBB2 PTPN11 (BIND; GRID; MINT) ERBB2 PTPN18 (HPRD) ERBB2
RASA1 (MINT)
ERBB2 RIN1 (MINT) ERBB2 RIN2 (MINT) ERBB2 SERPINA3 (GRID; HPRD)
ERBB2 SH2B2 (MINT) ERBB2 SH2B3 (MINT) ERBB2 SH2D1B (MINT) ERBB2
SH2D2A (MINT) ERBB2 SH3BGRL (GRID) ERBB2 SH3BGRL3 (BIND; MINT)
ERBB2 SH3BP2 (MINT) ERBB2 SHC1 (BIND; GRID; HPRD; MINT) ERBB2 SHC2
(MINT) ERBB2 SHC3 (MINT) ERBB2 SLA2 (MINT) ERBB2 SOCS1 (MINT) ERBB2
SOS1 (GRID; HPRD) ERBB2 SRC (BIND; GRID; HPRD) ERBB2 STAT1 (MINT)
ERBB2 STAT3 (MINT) ERBB2 STUB1 (GRID; HPRD) ERBB2 SUPT6H (MINT)
ERBB2 SYK (MINT) ERBB2 TAF1 (BIND) ERBB2 TEC (MINT) ERBB2 TGFA
(HPRD) ERBB2 TLN1 (MINT) ERBB2 TOB1 (GRID; HPRD) ERBB2 TP53RK
(BIND) ERBB2 TXK (MINT) ERBB2 UBB (HPRD) ERBB2 VAV2 (MINT) ERBB2
VAV3 (MINT) ERBB3 AGTR2 (HPRD) ERBB3 CD82 (CCSB; GRID; HPRD) ERBB3
CDK5 (HPRD) ERBB3 CHN2 (MINT) ERBB3 CRK (MINT) ERBB3 CRKL (MINT)
ERBB3 DAB1 (MINT) ERBB3 DAPP1 (MINT) ERBB3 EGF (GRID; HPRD) ERBB3
EGR1 (GRID) ERBB3 ERBB4 (HPRD; IntAct) ERBB3 FER (MINT) ERBB3 FGFR1
(MINT) ERBB3 FLYWCH1 (HPRD; MDC) ERBB3 GRB2 (BIND; GRID; HPRD)
ERBB3 GRB7 (CCSB; GRID; HPRD; MINT) ERBB3 HCK (MINT) ERBB3 HNF4A
(BIND) ERBB3 IL6ST (GRID; HPRD) ERBB3 ITK (MINT) ERBB3 JAK2 (MINT)
ERBB3 JAK3 (MINT) ERBB3 MUC1 (GRID: HPRD) ERBB3 NCK1 (MINT) ERBB3
NCK2 (MINT) ERBB3 NRG1 (BIND; CCSB; DIP; GRID; HPRD; MINT) ERBB3
NRG2 (GRID; HPRD) ERBB3 ODF2L (HPRD; MDC) ERBB3 PA2G4 (CCSB; GRID;
HPRD; IntAct) ERBB3 PIK3R1 (GRID; HPRD; IntAct; MINT) ERBB3 PIK3R2
(BIND; GRID; MINT) ERBB3 PLCG1 (MINT) ERBB3 PTGES3 (CCSB; GRID;
HPRD) ERBB3 PTK2 (CCSB; GRID; HPRD) ERBB3 PTK2B (HPRD) ERBB3 PTK6
(GRID; HPRD; MINT) ERBB3 RASA1 (MINT) ERBB3 RASA4 (HPRD; IntAct;
MDC; MINT) ERBB3 RGS4 (CCSB; GRID; HPRD) ERBB3 RIN1 (MINT) ERBB3
RNF41 (HPRD) ERBB3 RPN1 (GRID) ERBB3 SH2B3 (MINT) ERBB3 SH2D1A
(MINT) ERBB3 SHC1 (BIND; CCSB; GRID; HPRD; MINT) ERBB3 SHC3 (MINT)
ERBB3 SOS1 (GRID; HPRD; MINT) ERBB3 SRC (MINT) ERBB3 SYK (MINT)
ERBB3 TNS4 (MINT) ERBB3 TXK (MINT) ERBB3 VAV2 (MINT) ERBB3 VAV3
(MINT) ERBB3 ZAP70 (MINT) ERBB3 ZNF207 (GRID) ERBB4 ADAM17 (HPRD)
ERBB4 ANKS1A (MINT) ERBB4 BTC (GRID; HPRD) ERBB4 CD44 (GRID; HPRD)
ERBB4 CRK (BIND; GRID) ERBB4 CRKL (MINT) ERBB4 CTGF (GRID; HPRD)
ERBB4 DLG1 (GRID; HPRD) ERBB4 DLG2 (BIND; GRID; HPRD; IntAct) ERBB4
DLG3 (BIND; GRID; HPRD; IntAct) ERBB4 DLG4 (BIND; GRID; HPRD;
IntAct; MINT) ERBB4 EREG (GRID; HPRD) ERBB4 GRB2 (BIND; GRID) ERBB4
GRIN1 (IntAct) ERBB4 HBEGF (GRID; HPRD) ERBB4 MUC1 (GRID; HPRD)
ERBB4 NCK1 (GRID) ERBB4 NCK2 (BIND; MINT) ERBB4 NRG1 (DIP; GRID;
HPRD; MINT) ERBB4 NRG2 (GRID; HPRD) ERBB4 NRG3 (HPRD) ERBB4 NRG4
(HPRD) ERBB4 PIK3R2 (BIND; GRID; MINT) ERBB4 PTPN11 (BIND; GRID;
MINT) ERBB4 RASA1 (MINT) ERBB4 RNF41 (GRID; HPRD) ERBB4 SHC1 (BIND;
GRID; MINT) ERBB4 SNTB2 (BIND; GRID; HPRD; IntAct) ERBB4 STAT5A
(GRID; HPRD) ERBB4 STAT5B (BIND; MINT) ERBB4 SYK (MINT) ERBB4 TGFA
(HPRD) ERBB4 YAP1 (GRID; HPRD) FER CALM1 (HPRD) FER CTNNB1 (HPRD)
FER CTNND1 (GRID; HPRD) FER CTTN (GRID; HPRD) FER IRS1 (HPRD) FER
JAK1 (HPRD) FER JUP (HPRD) FER STAT3 (GRID; HPRD) FER TMF1 (GRID;
HPRD) FER YWHAB (HPRD) FES BCAR1 (GRID; HPRD) FES BCR (GRID; HPRD)
FES CSF2RB (HPRD) FES DOK1 (HPRD) FES DPYSL5 (CCSB; GRID; HPRD) FES
HSH2D (GRID; HPRD) FES IL4R (GRID; HPRD) FES IRS1 (HPRD) FES IRS2
(HPRD) FES JAK1 (HPRD) FES JAK2 (HPRD) FES JAK3 (HPRD) FES PIK3R1
(GRID; HPRD) FES PLXNA1 (HPRD) FES PSMD13 (GRID) FES RASA1 (CCSB;
GRID; HPRD) FES RASA3 (GRID; HPRD) FES STAT3 (CCSB; GRID; HPRD)
FGFR1 ATF2 (BIND) FGFR1 BNIP2 (BIND; CCSB; GRID; HPRD) FGFR1 CBL
(reactome) FGFR1 CREBBP (HPRD) FGFR1 CRK (HPRD) FGFR1 FDPS (CCSB;
GRID; HPRD) FGFR1 FGF1 (CCSB; DIP; GRID; HPRD; MINT; reactome)
FGFR1 FGF17 (HPRD; reactome) FGFR1 FGF18 (HPRD) FGFR1 FGF2 (DIP;
GRID; HPRD; IntAct; reactome) FGFR1 FGF20 (reactome) FGFR1 FGF23
(reactome) FGFR1 FGF3 (HPRD) FGFR1 FGF4 (HPRD; reactome) FGFR1 FGF5
(DIP; GRID; HPRD; reactome) FGFR1 FGF6 (HPRD; reactome) FGFR1 FGF7
(HPRD) FGFR1 FGF8 (HPRD; reactome) FGFR1 FGF9 (HPRD; reactome)
FGFR1 FGFR2 (BIND) FGFR1 FRS2 (CCSB; GRID; HPRD; reactome) FGFR1
FRS3 (BIND; GRID; HPRD; reactome) FGFR1 GRB14 (GRID; HPRD) FGFR1
GRB2 (CCSB; GRID; HPRD; reactome) FGFR1 IL17RD (HPRD) FGFR1 JUN
(BIND) FGFR1 KL (reactome) FGFR1 KPNB1 (GRID; HPRD) FGFR1 MMP2
(HPRD) FGFR1 NCAM1 (GRID; HPRD) FGFR1 NCK2 (BIND; HPRD) FGFR1 NRP1
(HPRD) FGFR1 PIK3R1 (HPRD) FGFR1 PIK3R2 (BIND; HPRD) FGFR1 PLCG1
(BIND; HPRD) FGFR1 RPS6KA1 (BIND; HPRD) FGFR1 RTN1 (BIND) FGFR1
RTN3 (BIND; HPRD) FGFR1 SH3BP2 (BIND; HPRD) FGFR1 SHB (BIND; GRID)
FGFR1 SHC1 (CCSB; GRID; HPRD) FGFR1 SLA (BIND; HPRD) FGFR1 SOS1
(HPRD) FGFR1 TENC1 (BIND; HPRD) FGFR2 CACNA1D (GRID; HPRD) FGFR2
CBL (reactome) FGFR2 FGF1 (DIP; GRID; HPRD; IntAct; MINT; reactome)
FGFR2 FGF10 (DIP; GRID; HPRD; IntAct; MINT; reactome) FGFR2 FGF16
(reactome) FGFR2 FGF17 (HPRD; reactome) FGFR2 FGF18 (HPRD;
reactome) FGFR2 FGF2 (DIP; GRID; IntAct; reactome) FGFR2 FGF20
(reactome) FGFR2 FGF22 (reactome) FGFR2 FGF23 (HPRD; reactome)
FGFR2 FGF3 (DIP; HPRD; reactome) FGFR2 FGF4 (HPRD; reactome) FGFR2
FGF5 (GRID; HPRD; reactome) FGFR2 FGF6 (DIP; HPRD; reactome) FGFR2
FGF7 (DIP; GRID; HPRD; reactome) FGFR2 FGF8 (HPRD; reactome) FGFR2
FGF9 (DIP; GRID; HPRD; reactome) FGFR2 FRS2 (reactome) FGFR2 FRS3
(reactome) FGFR2 FYN (HPRD) FGFR2 GRB2 (reactome) FGFR2 ITGA5
(HPRD) FGFR2 PAK4 (HPRD) FGFR2 PLCG1 (GRID; HPRD) FGFR2 UQCRB
(BIND) FGFR3 ATF3 (HPRD; IntAct; MDC) FGFR3 C13orf34 (HPRD; IntAct;
MDC) FGFR3 C6orf47 (GRID; HPRD) FGFR3 CBL (reactome) FGFR3 CCDC17
(HPRD; IntAct; MDC) FGFR3 CENTD2 (HPRD; IntAct; MDC) FGFR3 CHGB
(HPRD; IntAct; MDC) FGFR3 CTSK (HPRD; IntAct; MDC) FGFR3 FGF1 (DIP;
GRID; HPRD; IntAct; reactome) FGFR3 FGF16 (reactome) FGFR3 FGF17
(HPRD; reactome) FGFR3 FGF18 (HPRD; reactome) FGFR3 FGF2 (DIP;
HPRD; reactome) FGFR3 FGF20 (reactome) FGFR3 FGF23 (HPRD; reactome)
FGFR3 FGF3 (HPRD) FGFR3 FGF4 (DIP; HPRD; reactome) FGFR3 FGF5 (DIP;
HPRD; reactome) FGFR3 FGF6 (HPRD) FGFR3 FGF7 (DIP; HPRD) FGFR3 FGF8
(GRID; HPRD; reactome) FGFR3 FGF9 (GRID; HPRD; reactome) FGFR3
FGFR2 (BIND) FGFR3 FRS2 (reactome) FGFR3 FRS3 (reactome) FGFR3
GPSM3 (BIND; HPRD; IntAct; MINT) FGFR3 GRB2 (GRID; HPRD; reactome)
FGFR3 GTF3C1 (HPRD; MDC) FGFR3 HBZ (HPRD; IntAct; MDC) FGFR3 HNRNPL
(HPRD; IntAct; MDC) FGFR3 KIAA1377 (HPRD; IntAct; MDC) FGFR3 KRT8
(HPRD; IntAct; MDC) FGFR3 NDUFS6 (HPRD; IntAct; MDC)
FGFR3 POLA2 (HPRD; IntAct; MDC) FGFR3 PTPN11 (GRID) FGFR3 RADIL
(HPRD; MDC) FGFR3 RNF130 (HPRD; IntAct; MDC) FGFR3 RPL8 (HPRD;
IntAct; MDC) FGFR3 SH2B1 (GRID; HPRD) FGFR3 SLC25A6 (HPRD; IntAct;
MDC) FGFR3 SMA4 (IntAct) FGFR3 SMG7 (HPRD; MDC) FGFR3 STAT1 (HPRD)
FGFR3 STAT3 (HPRD) FGFR4 ADH6 (BIND) FGFR4 ATF2 (BIND) FGFR4 ATP5H
(BIND) FGFR4 CALM1 (BIND) FGFR4 CBL (reactome) FGFR4 CDH2 (HPRD)
FGFR4 COX6C (BIND) FGFR4 COX7B (BIND) FGFR4 DPM2 (BIND) FGFR4 E2F4
(BIND) FGFR4 EIF3E (BIND) FGFR4 FBP1 (BIND) FGFR4 FGA (BIND) FGFR4
FGF1 (DIP; GRID; HPRD; reactome) FGFR4 FGF16 (reactome) FGFR4 FGF17
(HPRD; reactome) FGFR4 FGF18 (HPRD; reactome) FGFR4 FGF19 (BIND;
DIP; GRID; HPRD; reactome) FGFR4 FGF2 (DIP; GRID; HPRD; reactome)
FGFR4 FGF20 (reactome) FGFR4 FGF23 (reactome) FGFR4 FGF3 (HPRD)
FGFR4 FGF4 (HPRD; reactome) FGFR4 FGF5 (HPRD) FGFR4 FGF6 (GRID;
HPRD; reactome) FGFR4 FGF7 (HPRD) FGFR4 FGF8 (GRID; HPRD; reactome)
FGFR4 FGF9 (HPRD; reactome) FGFR4 FN1 (BIND) FGFR4 FRS2 (reactome)
FGFR4 FRS3 (reactome) FGFR4 GJC1 (BIND) FGFR4 GLRX (BIND) FGFR4 GPT
(BIND) FGFR4 GRB2 (reactome) FGFR4 GSTO1 (BIND) FGFR4 HNRNPC (BIND)
FGFR4 LDLRAP1 (BIND) FGFR4 PLCG1 (HPRD) FGFR4 POLR2I (BIND) FGFR4
PPIA (BIND) FGFR4 PSMA4 (BIND) FGFR4 RPLP2 (BIND) FGFR4 SF3B4
(BIND) FGFR4 STAT1 (HPRD) FGFR4 STAT3 (HPRD) FGFR4 TLR4 (BIND) FGR
ARRB1 (GRID; HPRD) FGR CBL (GRID; HPRD) FGR CCR3 (GRID; HPRD) FGR
CD24 (GRID; HPRD) FGR DAB2 (GRID; HPRD) FGR DOK1 (HPRD) FGR HCLS1
(HPRD) FGR HSP90AA1 (DIP) FGR INPP5D (GRID; HPRD) FGR NCOA6 (GRID)
FGR PLAUR (DIP) FGR PTK2 (GRID; HPRD) FGR SLAMF1 (GRID; HPRD) FGR
SNCA (HPRD) FGR SRC (HPRD) FGR SYK (GRID; HPRD; MINT) FGR VDR
(HPRD) FGR WAS (GRID; HPRD; MINT) FGR YWHAQ (GRID; HPRD) FLT1 ATR
(GRID; HPRD) FLT1 CRK (HPRD) FLT1 CTNNB1 (HPRD) FLT1 FYN (HPRD)
FLT1 GRB2 (HPRD) FLT1 KDR (GRID; HPRD) FLT1 NCK1 (BIND; HPRD) FLT1
NRP1 (HPRD) FLT1 NRP2 (HPRD) FLT1 PGF (DIP; GRID; HPRD; IntAct)
FLT1 PIK3R1 (BIND) FLT1 PLCG1 (BIND; HPRD) FLT1 PLCG2 (GRID; HPRD)
FLT1 PTK2 (HPRD) FLT1 PTPN11 (BIND; HPRD) FLT1 SHC1 (GRID; HPRD)
FLT1 SHC2 (MINT) FLT1 STAT1 (HPRD) FLT1 STAT3 (HPRD) FLT1 VEGFA
(BIND; DIP; GRID; HPRD; IntAct) FLT1 VEGFB (GRID; HPRD) FLT1 YES1
(HPRD) FLT3 CBLB (GRID; HPRD) FLT3 FIZ1 (HPRD) FLT3 FLT3LG (GRID;
HPRD) FLT3 GRB2 (GRID; HPRD) FLT3 INPP5D (HPRD) FLT3 NCK1 (HPRD)
FLT3 PTPN12 (HPRD) FLT3 PTPN6 (HPRD) FLT3 SH3BP2 (HPRD) FLT3 SHC1
(GRID; HPRD) FLT3 SOCS1 (GRID; HPRD) FLT4 ATF7IP (BIND; HPRD) FLT4
FIGF (DIP; GRID; HPRD) FLT4 GRB2 (GRID; HPRD) FLT4 ITGA5 (HPRD)
FLT4 ITGB1 (GRID; HPRD) FLT4 KDR (HPRD; IntAct) FLT4 PTK2 (HPRD)
FLT4 PTPN11 (HPRD) FLT4 SHC1 (GRID; HPRD) FLT4 SHC3 (HPRD) FLT4
VEGFC (BIND; DIP; GRID; HPRD) FRK HNF4A (BIND) FRK RB1 (BIND; CCSB;
GRID; HPRD) FYN ACP1 (HPRD) FYN ADAM15 (CCSB; GRID; HPRD; MINT) FYN
ADD2 (GRID; HPRD) FYN ATXN1 (HPRD; IntAct; MINT) FYN BCAR1 (GRID;
HPRD) FYN BCL3 (GRID; HPRD) FYN CASP3 (HPRD) FYN CASP8 (HPRD) FYN
CAV1 (HPRD) FYN CBL (BIND; GRID; HPRD; MINT) FYN CBLB (HPRD) FYN
CD19 (GRID; HPRD) FYN CD2 (BIND; CCSB; GRID; HPRD; MINT) FYN CD226
(GRID; HPRD) FYN CD247 (BIND; CCSB; GRID; HPRD) FYN CD2AP (GRID;
HPRD; MINT) FYN CD36 (GRID; HPRD) FYN CD44 (CCSB; GRID; HPRD) FYN
CD48 (CCSB; GRID) FYN CD5 (HPRD) FYN CD55 (CCSB; GRID; HPRD) FYN
CD79A (HPRD) FYN CD79B (HPRD) FYN CDC2 (CCSB; GRID; HPRD) FYN CDK5
(HPRD) FYN CNN1 (HPRD) FYN CNN3 (HPRD) FYN CNTN1 (GRID; HPRD) FYN
CNTNAP1 (HPRD) FYN CRK (HPRD) FYN CSF2RB (HPRD) FYN CTLA4 (GRID;
HPRD) FYN CTNNB1 (HPRD) FYN CTNND1 (GRID) FYN CTNND2 (HPRD) FYN DCC
(BIND) FYN DLG4 (GRID; HPRD) FYN DOK1 (HPRD) FYN DOK3 (HPRD) FYN
DOK4 (BIND; HPRD) FYN EVL (CCSB; GRID; HPRD) FYN FAS (CCSB; GRID;
HPRD) FYN FASLG (GRID; HPRD) FYN FCER2 (BIND; CCSB; GRID; HPRD) FYN
FCGR2A (HPRD) FYN FLOT1 (HPRD) FYN FLOT2 (HPRD) FYN FN1 (HPRD) FYN
FNBP4 (CCSB; GRID; HPRD) FYN FYB (GRID; HPRD; MINT) FYN GAB3 (GRID;
HPRD) FYN GNB2L1 (CCSB; GRID; HPRD) FYN GP6 (GRID; HPRD) FYN GRAP
(HPRD) FYN GRB10 (GRID; HPRD) FYN GRIN2A (GRID; HPRD) FYN GRIN2B
(GRID; HPRD) FYN HNRPK (BIND; CCSB; GRID; HPRD) FYN HRAS (HPRD;
MINT) FYN HTR6 (IntAct) FYN IL2RB (HPRD) FYN IL7R (BIND; GRID;
HPRD) FYN IRS1 (HPRD) FYN ITGAV (HPRD) FYN ITGB4 (HPRD) FYN ITGB6
(HPRD) FYN ITK (GRID; HPRD) FYN ITPR1 (HPRD) FYN JAK2 (GRID; HPRD)
FYN JUP (HPRD) FYN KDR (HPRD; MINT) FYN KHDRBS1 (GRID; HPRD;
IntAct; MINT) FYN KIT (HPRD) FYN LCP2 (HPRD) FYN LYN (HPRD) FYN MAG
(GRID; HPRD) FYN MAP2 (HPRD; MINT) FYN MAPT (HPRD) FYN MCAM (GRID;
HPRD) FYN MS4A1 (CCSB; GRID; HPRD) FYN NCAM1 (GRID; HPRD) FYN NCK1
(MINT) FYN NEDD9 (GRID; HPRD) FYN NMT1 (HPRD) FYN NPHS1 (GRID;
HPRD) FYN NTRK2 (GRID; HPRD) FYN PAG1 (GRID; HPRD) FYN PDE4D (BIND)
FYN PDGFRB (CCSB; GRID; HPRD) FYN PECAM1 (GRID; HPRD) FYN PIK3R1
(BIND; GRID; HPRD) FYN PIK3R2 (GRID; HPRD) FYN PLAUR (CCSB; GRID;
HPRD) FYN PLCG1 (GRID; HPRD) FYN PLCG2 (CCSB; GRID; HPRD) FYN PLD2
(GRID; HPRD) FYN PRKCD (HPRD; IntAct) FYN PRKCE (HPRD) FYN PRKCH
(HPRD) FYN PRKCQ (GRID; HPRD) FYN PRKCZ (HPRD) FYN PTK2 (GRID;
HPRD) FYN PTPN11 (CCSB; GRID; HPRD) FYN PTPN5 (GRID; HPRD) FYN
PTPRA (CCSB; GRID; HPRD) FYN PTPRC (GRID; HPRD) FYN PTPRE (HPRD)
FYN PTPRF (HPRD) FYN PTPRZ1 (HPRD) FYN PXN (GRID; HPRD) FYN RAF1
(CCSB; GRID; HPRD) FYN RICS (GRID; HPRD) FYN SDC3 (CCSB; GRID;
HPRD) FYN SH2B2 (HPRD) FYN SH2D1A (GRID; HPRD; MINT) FYN SH3BP2
(HPRD) FYN SHC1 (HPRD; MINT) FYN SIT1 (GRID; HPRD) FYN SKAP1 (GRID;
HPRD) FYN SKAP2 (CCSB; GRID; HPRD) FYN SLAMF1 (CCSB; GRID; HPRD)
FYN SNCA (GRID; HPRD) FYN SNX26 (HPRD) FYN SOCS1 (BIND; GRID; HPRD)
FYN SOS1 (GRID; HPRD) FYN SPHK1 (IntAct) FYN SPHK2 (IntAct) FYN SPN
(GRID; HPRD)
FYN SRC (MINT) FYN STAT1 (HPRD) FYN SYK (CCSB; GRID; HPRD) FYN TEK
(HPRD) FYN THY1 (CCSB; GRID; HPRD) FYN TNK2 (CCSB; GRID; HPRD) FYN
TOM1L1 (HPRD) FYN TRAT1 (HPRD) FYN TRPC6 (GRID; HPRD) FYN TRPV4
(GRID; HPRD) FYN TUBA1B (CCSB; GRID; HPRD) FYN TUBA3C (CCSB; GRID;
HPRD) FYN TUBA4A (CCSB; GRID; HPRD) FYN TXK (HPRD) FYN TYK2 (CCSB;
GRID; HPRD) FYN TYRO3 (GRID; HPRD) FYN UNC119 (CCSB; GRID; HPRD)
FYN VAV1 (CCSB; GRID; HPRD) FYN WAS (CCSB; GRID; HPRD; MINT) FYN
WASF1 (GRID; HPRD) FYN WASF2 (GRID; HPRD) FYN YTHDC1 (GRID; HPRD)
FYN ZAP70 (CCSB; GRID; HPRD) HCK ACTB (HPRD) HCK ADAM15 (CCSB;
GRID; HPRD; MINT) HCK AGK (HPRD; MDC) HCK ARRB1 (HPRD) HCK BCAR1
(GRID; HPRD) HCK BCR (GRID; HPRD) HCK C14orf4 (HPRD) HCK C2orf44
(HPRD; IntAct) HCK CBL (GRID; HPRD) HCK CCR3 (GRID; HPRD) HCK CD2AP
(HPRD; IntAct) HCK CSF2RB (HPRD) HCK CSF3R (GRID; HPRD) HCK DDEF1
(HPRD; IntAct) HCK DNM2 (HPRD; IntAct) HCK DOK1 (HPRD) HCK ELMO1
(GRID; HPRD; IntAct; MINT) HCK EVL (HPRD; IntAct) HCK FCGR1A (CCSB;
GRID; HPRD) HCK FCGR2A (CCSB; GRID; HPRD) HCK GALNAC4S-6ST (GRID;
HPRD) HCK HNRPK (HPRD) HCK IL6ST (GRID; HPRD) HCK INPP5D (HPRD) HCK
KHDRBS1 (BIND; HPRD; IntAct) HCK KIT (HPRD) HCK LCP2 (HPRD; IntAct)
HCK PECAM1 (GRID; HPRD) HCK PIK3CB (HPRD) HCK PIK3R1 (HPRD; IntAct)
HCK PIK3R2 (HPRD; IntAct) HCK PLAUR (CCSB; DIP; GRID; HPRD) HCK
PLCG1 (GRID; HPRD) HCK PLCG2 (HPRD) HCK RAPGEF1 (GRID; HPRD) HCK
RASA1 (CCSB; GRID; HPRD) HCK RASA3 (GRID; HPRD) HCK SF3B3 (HPRD;
IntAct) HCK SH3BP1 (HPRD; IntAct) HCK SH3KBP1 (HPRD; IntAct) HCK
SKAP2 (CCSB; GRID; HPRD) HCK SOS1 (HPRD; IntAct) HCK STAT3 (HPRD)
HCK TRPV4 (GRID; HPRD) HCK TSG101 (HPRD; IntAct) HCK UNC119 (CCSB;
GRID; HPRD) HCK VAV1 (HPRD) HCK WAS (HPRD; IntAct; MINT) HCK WIPF1
(CCSB; GRID; HPRD; IntAct; MINT) IGF1R ARHGEF12 (GRID; HPRD; MINT)
IGF1R CRK (HPRD) IGF1R CRKL (HPRD) IGF1R DOK4 (HPRD) IGF1R DOK5
(HPRD) IGF1R EHD1 (BIND; GRID; HPRD) IGF1R ESR1 (HPRD) IGF1R GIGYF1
(GRID; HPRD) IGF1R GIPC1 (HPRD) IGF1R GNAI1 (GRID; HPRD) IGF1R
GNAI2 (GRID; HPRD) IGF1R GNB2L1 (BIND; GRID; HPRD) IGF1R GRB10
(BIND; GRID; HPRD; MINT) IGF1R GYS1 (BIND) IGF1R HNF1A (BIND) IGF1R
IGF1 (BIND; DIP; GRID; HPRD) IGF1R IGF2 (GRID; HPRD) IGF1R IGFBP3
(GRID; HPRD) IGF1R INS (BIND; GRID; HPRD) IGF1R INSR (HPRD) IGF1R
IRS1 (BIND; GRID; HPRD) IGF1R IRS2 (GRID; HPRD; MINT) IGF1R IRS4
(HPRD) IGF1R ITGB1 (BIND; HPRD) IGF1R JAK1 (BIND; GRID; HPRD) IGF1R
JAK2 (HPRD) IGF1R KRT27 (HPRD) IGF1R MAP3K5 (HPRD) IGF1R MDM2
(HPRD) IGF1R NEDD4 (GRID; HPRD) IGF1R PBEF1 (BIND) IGF1R PIK3R1
(DIP; GRID; HPRD) IGF1R PIK3R2 (BIND; HPRD) IGF1R PIK3R3 (GRID;
HPRD) IGF1R PRKCD (BIND; HPRD) IGF1R PRKD1 (BIND; HPRD) IGF1R PTPN1
(GRID; HPRD) IGF1R PTPN11 (DIP; GRID; HPRD) IGF1R RASA1 (GRID;
HPRD) IGF1R SHC1 (BIND; DIP; GRID; HPRD; IntAct) IGF1R SNAP29
(GRID) IGF1R SOCS1 (BIND; GRID; HPRD; MINT) IGF1R SOCS2 (BIND;
GRID; HPRD; MINT) IGF1R SOCS3 (BIND; GRID; HPRD; MINT) IGF1R SRC
(HPRD) IGF1R STAT3 (HPRD) IGF1R TAF1 (BIND) IGF1R VAV3 (GRID: HPRD)
IGF1R WISP2 (HPRD) IGF1R YWHAB (BIND; GRID; HPRD; IntAct; MINT)
IGF1R YWHAE (BIND; GRID; HPRD; MINT) IGF1R YWHAG (GRID; HPRD; MINT)
IGF1R YWHAZ (BIND; GRID; HPRD) INSR ACP1 (HPRD) INSR ADRB2 (HPRD)
INSR AHSG (GRID; HPRD) INSR ARF1 (BIND; HPRD) INSR ARHGAP26 (HPRD)
INSR CALM1 (HPRD) INSR CALM2 (HPRD) INSR CALM3 (HPRD) INSR CASP7
(BIND) INSR CAV1 (GRID; HPRD) INSR CAV3 (HPRD) INSR CBL (HPRD) INSR
CEACAM1 (HPRD) INSR CRK (HPRD) INSR CRKL (HPRD; IntAct) INSR DOK1
(HPRD; reactome) INSR DOK4 (HPRD) INSR DOK5 (HPRD) INSR ENPP1
(GRID; HPRD) INSR FABP4 (HPRD) INSR FRS2 (GRID; HPRD) INSR GAB1
(GRID; HPRD) INSR GNB2L1 (HPRD) INSR GRB10 (BIND; GRID; HPRD; MINT;
reactome) INSR GRB14 (GRID; HPRD; MINT) INSR GRB2 (reactome) INSR
GRB7 (GRID; HPRD) INSR HMGA1 (BIND) INSR HNF1A (BIND) INSR HRAS
(GRID; HPRD) INSR IGF2 (GRID; HPRD) INSR IMPDH2 (BIND) INSR INS
(DIP; GRID; HPRD; reactome) INSR INSRR (GRID; HPRD) INSR IRS1
(BIND; DIP; GRID; HPRD; IntAct; MINT; reactome) INSR IRS2 (GRID;
HPRD; MINT; reactome) INSR JAK1 (BIND; HPRD) INSR JAK2 (HPRD) INSR
KHDRBS1 (HPRD) INSR KRT27 (HPRD) INSR MAD2L1 (HPRD; MINT) INSR
MAPK3 (GRID; HPRD) INSR PBEF1 (BIND) INSR PIK3CA (reactome) INSR
PIK3CB (reactome) INSR PIK3R1 (BIND; DIP; HPRD; reactome) INSR
PIK3R2 (reactome) INSR PIK3R3 (GRID; HPRD) INSR PLCG1 (HPRD) INSR
PRKCA (HPRD) INSR PRKCD (GRID; HPRD) INSR PTK2 (HPRD) INSR PTPN1
(GRID; HPRD; MINT) INSR PTPN11 (DIP; GRID; HPRD) INSR PTPN12 (GRID;
HPRD) INSR PTPN2 (HPRD) INSR PTPN6 (GRID; HPRD) INSR PTPRC (HPRD)
INSR PTPRF (GRID; HPRD) INSR RAF1 (GRID; HPRD) INSR RASA1 (GRID;
HPRD) INSR SH2B1 (GRID; HPRD) INSR SH2B2 (HPRD) INSR SHC1 (BIND;
DIP; GRID; HPRD; IntAct; reactome) INSR SMAD2 (GRID; HPRD) INSR
SNX1 (GRID; HPRD) INSR SNX2 (GRID; HPRD) INSR SNX4 (GRID; HPRD)
INSR SNX6 (GRID; HPRD) INSR SOCS1 (BIND; HPRD) INSR SOCS2 (HPRD)
INSR SOCS3 (GRID; HPRD) INSR SOCS6 (BIND; HPRD) INSR SORBS1 (GRID;
HPRD) INSR SOS1 (reactome) INSR SRC (HPRD) INSR STAT5A (HPRD) INSR
STAT5B (HPRD) INSR SYNCRIP (GRID; HPRD) INSR VAV1 (GRID; HPRD) INSR
VAV3 (GRID; HPRD) INSR YWHAB (HPRD; IntAct) INSRR KRT27 (HPRD) ITK
BLNK (GRID; HPRD) ITK CBL (GRID; HPRD) ITK CD28 (GRID; HPRD) ITK
GRB2 (GRID; HPRD) ITK HNRPK (BIND; GRID; HPRD) ITK KHDRBS1 (GRID;
HPRD) ITK KPNA2 (BIND; GRID; HPRD) ITK LAT (BIND; GRID; HPRD) ITK
LCK (HPRD) ITK LCP2 (BIND; GRID; HPRD; MINT) ITK PLCG1 (BIND; GRID;
HPRD) ITK PLCG2 (HPRD) ITK PPIA (GRID; HPRD) ITK SH2D2A (HPRD) ITK
SMAD4 (BIND; HPRD) ITK SOCS1 (BIND; GRID; HPRD) ITK TGFBR1 (BIND;
HPRD) ITK WAS (GRID; HPRD) JAK1 ARRB1 (GRID) JAK1 ATIC (GRID) JAK1
BRCA1 (GRID; HPRD; MINT) JAK1 CCR1 (HPRD) JAK1 CCR5 (HPRD) JAK1
CSF2RB (HPRD) JAK1 CSF3R (GRID; HPRD) JAK1 CXCR4 (HPRD) JAK1 ELF3
(GRID) JAK1 ELP2 (GRID; HPRD) JAK1 GHR (GRID; HPRD) JAK1 GNB2L1
(BIND; GRID; HPRD; MINT) JAK1 GRB2 (DIP; GRID; HPRD) JAK1 IFNAR1
(DIP) JAK1 IFNAR2 (BIND; HPRD; MINT) JAK1 IFNGR1 (GRID; HPRD) JAK1
IL10RA (BIND; GRID; HPRD) JAK1 IL21R (GRID; HPRD) JAK1 IL2RB (BIND;
DIP; GRID; HPRD) JAK1 IL2RG (HPRD) JAK1 IL3RA (HPRD) JAK1 IL4R
(BIND; GRID; HPRD) JAK1 IL5RA (GRID; HPRD) JAK1 IL6R (MINT) JAK1
IL6ST (DIP; GRID; HPRD)
JAK1 IL7R (HPRD) JAK1 IL9R (GRID; HPRD) JAK1 INPP5D (HPRD) JAK1
IRS1 (HPRD) JAK1 IRS2 (GRID; HPRD) JAK1 JAK3 (GRID; HPRD) JAK1
JAKMIP1 (BIND; HPRD) JAK1 LRPPRC (MINT) JAK1 MDK (GRID; HPRD) JAK1
OSMR (GRID; HPRD) JAK1 PDGFRA (HPRD) JAK1 PDGFRB (HPRD) JAK1 PIK3R1
(GRID; HPRD) JAK1 PLA2G4A (HPRD) JAK1 PLAUR (BIND; DIP; GRID; HPRD)
JAK1 PLCG2 (HPRD) JAK1 PRKCZ (HPRD) JAK1 PRMT5 (GRID; HPRD) JAK1
PTPN11 (GRID; HPRD) JAK1 PTPN2 (HPRD) JAK1 PTPN6 (HPRD) JAK1 PTPRC
(HPRD) JAK1 RAF1 (GRID; HPRD) JAK1 SH2B2 (HPRD) JAK1 SHB (GRID;
HPRD) JAK1 SOCS1 (GRID; HPRD) JAK1 SOCS3 (HPRD) JAK1 STAM (HPRD)
JAK1 STAM2 (GRID; HPRD; MINT) JAK1 STAT1 (GRID; HPRD) JAK1 STAT2
(HPRD) JAK1 STAT3 (GRID; HPRD; MINT) JAK1 STAT5A (GRID HPRD) JAK1
STAT6 (HPRD) JAK1 SYK (GRID; HPRD) JAK1 TAF1 (BIND) JAK1 TEC (GRID;
HPRD) JAK1 TNFRSF1A (GRID; HPRD) JAK1 TSHR (GRID; HPRD) JAK1 TYK2
(GRID; HPRD) JAK2 AGTR1 (GRID; HPRD) JAK2 BCR (GRID; HPRD) JAK2
BRCA1 (GRID; HPRD, MINT) JAK2 CCR2 (GRID; HPRD) JAK2 CCR5 (GRID;
HPRD) JAK2 CRLF2 (HPRD) JAK2 CSF2RB (GRID; HPRD) JAK2 CSF3R (GRID;
HPRD) JAK2 CTLA4 (BIND; GRID, HPRD) JAK2 CXCR4 (GRID; HPRD) JAK2
DNAJA3 (BIND; GRID, HPRD) JAK2 ELP2 (GRID; HPRD) JAK2 EPOR (GRID;
HPRD) JAK2 GHR (GRID; HPRD) JAK2 GRB10 (GRID; HPRD) JAK2 GRB2
(GRID; HPRD) JAK2 GTF2I (HPRD) JAK2 HES1 (HPRD) JAK2 HES5 (HPRD)
JAK2 HSPA8 (GRID; HPRD) JAK2 HTR2A (GRID; HPRD) JAK2 IFNGR1 (HPRD)
JAK2 IFNGR2 (GRID; HPRD) JAK2 IL12RB2 (BIND; GRID; HPRD) JAK2 IL23R
(HPRD) JAK2 IL3RA (HPRD) JAK2 IL4R (HPRD) JAK2 IL5RA (GRID; HPRD)
JAK2 IRS1 (HPRD) JAK2 IRS2 (HPRD) JAK2 JAK3 (HPRD) JAK2 KIT (GRID;
HPRD) JAK2 LEPR (GRID; HPRD; IntAct) JAK2 LYN (GRID; HPRD) JAK2 MDK
(GRID; HPRD) JAK2 MPL (HPRD) JAK2 MST1R (HPRD) JAK2 NFKBIA (HPRD)
JAK2 OSMR (GRID; HPRD) JAK2 PDGFRB (HPRD) JAK2 PIK3R1 (GRID; HPRD)
JAK2 PKD1 (HPRD) JAK2 PLCG2 (HPRD) JAK2 PPIA (GRID; HPRD) JAK2
PPP2CA (HPRD) JAK2 PPP2R1B (GRID; HPRD) JAK2 PPP2R5A (GRID; HPRD)
JAK2 PRLR (GRID; HPRD) JAK2 PRMT5 (GRID; HPRD) JAK2 PTK2 (GRID;
HPRD) JAK2 PTPN1 (GRID; HPRD) JAK2 PTPN11 (GRID; HPRD) JAK2 PTPN12
(GRID; HPRD) JAK2 PTPN6 (GRID; HPRD) JAK2 PTPRC (HPRD) JAK2 RAF1
(GRID; HPRD) JAK2 SH2B1 (GRID; HPRD) JAK2 SH2B2 (HPRD) JAK2 SHC1
(GRID; HPRD) JAK2 SIRPA (GRID; HPRD) JAK2 SOCS1 (GRID; HPRD) JAK2
SOCS3 (GRID; HPRD) JAK2 STAM (GRID; HPRD) JAK2 STAM2 (GRID; HPRD)
JAK2 STAP2 (HPRD) JAK2 STAT1 (HPRD) JAK2 STAT2 (HPRD) JAK2 STAT3
(GRID; HPRD) JAK2 STAT5A (GRID; HPRD) JAK2 STAT5B (HPRD) JAK2 TEC
(GRID; HPRD) JAK2 TNFRSF1A (GRID; HPRD) JAK2 TSHR (GRID; HPRD) JAK2
TUB (GRID; HPRD) JAK2 TYK2 (HPRD) JAK2 UBASH3B (HPRD) JAK2 VAV1
(GRID; HPRD) JAK2 VCP (GRID; HPRD) JAK2 YES1 (GRID; HPRD) JAK3
CD247 (CCSB; GRID; HPRD) JAK3 CD40 (HPRD) JAK3 CXCR4 (CCSB; GRID;
HPRD) JAK3 IL2RB (CCSB; GRID; HPRD) JAK3 IL2RG (CCSB; DIP; GRID;
HPRD) JAK3 IL4R (GRID; HPRD) JAK3 IL6ST (HPRD) JAK3 IL7R (HPRD)
JAK3 INPP5D (HPRD) JAK3 IRS1 (GRID; HPRD) JAK3 IRS2 (GRID; HPRD)
JAK3 KHDRBS1 (HPRD; IntAct) JAK3 LCK (CCSB; GRID; HPRD) JAK3 PIK3R1
(HPRD) JAK3 PRMT5 (GRID; HPRD) JAK3 PTPN2 (HPRD) JAK3 PTPN6 (HPRD)
JAK3 PTPRC (GRID; HPRD) JAK3 SH2B2 (HPRD) JAK3 SHB (GRID; HPRD)
JAK3 SOCS1 (GRID; HPRD) JAK3 SOCS3 (HPRD) JAK3 STAM (CCSB; GRID;
HPRD) JAK3 STAM2 (CCSB; GRID; HPRD) JAK3 STAT3 (CCSB; GRID; HPRD)
JAK3 STAT5A (CCSB; GRID; HPRD) JAK3 TIAF1 (GRID; HPRD) KDR ACP1
(GRID; HPRD) KDR ANXA5 (GRID; HPRD) KDR ATR (HPRD) KDR CAV1 (HPRD)
KDR CBL (HPRD) KDR CDH5 (GRID; HPRD; MINT) KDR COL18A1 (GRID; HPRD)
KDR CRK (HPRD) KDR CSF2RB (HPRD) KDR CTNNB1 (HPRD) KDR DNM2 (HPRD)
KDR FIGF (DIP; GRID; HPRD) KDR FRS2 (HPRD) KDR GNA11 (HPRD) KDR
GNAQ (HPRD) KDR GRB10 (GRID; HPRD) KDR GRB2 (GRID; HPRD) KDR
HSP90AA1 (HPRD) KDR IQGAP1 (HPRD) KDR ITGB3 (HPRD) KDR NCK1 (HPRD;
MINT) KDR NRP1 (HPRD; MINT) KDR P2RY2 (HPRD) KDR PLCG1 (BIND; HPRD)
KDR PLCG2 (GRID; HPRD) KDR PLXNA1 (GRID) KDR PTPN11 (HPRD) KDR
PTPN6 (HPRD) KDR RASA1 (HPRD) KDR SH2D2A (BIND; HPRD) KDR SHB
(GRID; HPRD) KDR SHC1 (GRID; HPRD; MINT) KDR SHC2 (BIND; GRID;
HPRD; MINT) KDR SRC (HPRD) KDR STAT1 (HPRD) KDR SYNGAP1 (HPRD) KDR
TIMP3 (HPRD) KDR VEGFA (BIND; DIP; GRID; HPRD; MINT) KDR VEGFC
(BIND; GRID; HPRD) KDR YES1 (HPRD) KIT BCR (GRID) KIT CBL (HPRD)
KIT CBLB (HPRD) KIT CD63 (GRID) KIT CD81 (GRID; HPRD) KIT CD9
(GRID; HPRD) KIT CISH (HPRD) KIT CLTC (GRID; HPRD) KIT CRK (HPRD)
KIT CRKL (GRID; HPRD) KIT CSF2RA (HPRD) KIT CSF2RB (HPRD) KIT DOK1
(GRID; HPRD) KIT EPOR (GRID; HPRD) KIT GRAP (GRID; HPRD) KIT GRAP2
(GRID; HPRD) KIT GRB10 (GRID; HPRD) KIT GRB2 (BIND; DIP; GRID;
HPRD) KIT GRB7 (GRID; HPRD) KIT INPP5D (HPRD) KIT KITLG (GRID;
HPRD) KIT LCK (GRID; HPRD) KIT LYN (BIND; GRID; HPRD) KIT MATK
(GRID; HPRD) KIT MPDZ (GRID; HPRD) KIT PIK3CG (HPRD) KIT PIK3R1
(BIND; GRID; HPRD) KIT PIK3R2 (BIND; GRID; HPRD) KIT PLCE1 (HPRD)
KIT PLCG1 (BIND; GRID; HPRD) KIT PRKCA (HPRD) KIT PRKCB1 (HPRD) KIT
PTPN11 (BIND; GRID; HPRD) KIT PTPN6 (GRID; HPRD) KIT PTPRO (GRID;
HPRD) KIT PTPRU (HPRD) KIT RASA1 (HPRD) KIT SH2B2 (HPRD) KIT SH2B3
(HPRD) KIT SOCS1 (BIND; GRID; HPRD) KIT SOCS5 (HPRD) KIT SOCS6
(HPRD) KIT SPRED1 (HPRD) KIT SPRED2 (HPRD) KIT SRC (HPRD) KIT STAP1
(HPRD) KIT STAT1 (BIND; GRID; HPRD) KIT STAT5A (HPRD) KIT STAT5B
(HPRD) KIT TEC (GRID; HPRD) KIT YES1 (HPRD) LCK ACP1 (HPRD) LCK
ADAM15 (CCSB; GRID; HPRD; MINT) LCK AP2A1 (reactome) LCK AP2A2
(reactome) LCK AP2B1 (reactome) LCK AP2M1 (reactome) LCK AP2S1
(reactome) LCK ATP6V1H (reactome) LCK BCAR1 (GRID; HPRD) LCK CAMLG
(BIND) LCK CBL (GRID; HPRD; MINT) LCK CCR5 (GRID; HPRD) LCK CD2
(CCSB; GRID; HPRD; MINT) LCK CD247 (HPRD; MINT)
LCK CD28 (GRID; HPRD) LCK CD38 (BIND; CCSB; GRID; HPRD) LCK CD3E
(GRID; HPRD) LCK CD4 (CCSB; GRID; HPRD; MINT; reactome) LCK CD44
(CCSB; GRID; HPRD) LCK CD48 (CCSB; GRID; HPRD) LCK CD5 (DIP; GRID;
HPRD) LCK CD55 (CCSB; GRID; HPRD) LCK CD79A (HPRD) LCK CD79B (HPRD)
LCK CD8A (HPRD) LCK CD8B (BIND) LCK CDC25C (CCSB; GRID; HPRD) LCK
CSF2RB (HPRD) LCK CSF3R (HPRD) LCK CTLA4 (GRID; HPRD) LCK CTNND1
(GRID) LCK CTNND2 (GRID; HPRD) LCK DAPP1 (HPRD) LCK DEF6 (HPRD) LCK
DLG1 (GRID; HPRD) LCK DNM2 (BIND) LCK DOK1 (HPRD) LCK DOK2 (CCSB;
GRID; HPRD) LCK DOK3 (HPRD) LCK ESR1 (HPRD) LCK ESR2 (HPRD) LCK EZR
(HPRD) LCK FAS (CCSB; GRID; HPRD) LCK FASLG (GRID; HPRD) LCK FCGR3A
(CCSB; GRID; HPRD) LCK GAB2 (HPRD) LCK GRAP (HPRD) LCK HSP90AA1
(DIP; MINT) LCK IFNAR1 (HPRD) LCK IL2RB (CCSB; GRID; HPRD) LCK
KHDRBS1 (BIND; GRID; HPRD; IntAct; MINT) LCK KIR2DL3 (HPRD) LCK LAT
(HPRD) LCK LAX1 (HPRD) LCK LCP2 (HPRD; MINT) LCK LIME1 (HPRD) LCK
LYN (HPRD) LCK MAPK1 (HPRD; MINT) LCK MAPK3 (HPRD) LCK MS4A1 (HPRD)
LCK MUC1 (HPRD) LCK NEDD9 (GRID; HPRD) LCK NFKBIA (BIND; HPRD) LCK
NOTCH1 (GRID; HPRD) LCK NR3C1 (MINT) LCK NXF1 (IntAct) LCK PAG1
(GRID; HPRD) LCK PECAM1 (GRID; HPRD) LCK PIK3CA (GRID; HPRD) LCK
PIK3R1 (GRID; HPRD; MINT) LCK PLCG1 (GRID; HPRD) LCK PLCG2 (HPRD)
LCK PLD2 (GRID; HPRD) LCK PRKACA (HPRD) LCK PRKCA (HPRD) LCK PRKCD
(HPRD) LCK PRKCQ (HPRD) LCK PTK2 (HPRD) LCK PTPN11 (CCSB; GRID;
HPRD) LCK PTPN6 (CCSB; GRID; HPRD) LCK PTPRC (GRID; HPRD; IntAct)
LCK PTPRF (HPRD) LCK PTPRH (HPRD) LCK PXN (GRID; HPRD) LCK RAF1
(CCSB; GRID; HPRD) LCK RASA1 (HPRD) LCK SH2B3 (CCSB; GRID; HPRD)
LCK SH2D1A (HPRD; MINT) LCK SH2D2A (HPRD; IntAct) LCK SH3BP2 (HPRD)
LCK SHC1 (HPRD) LCK SIT1 (GRID; HPRD) LCK SKAP1 (GRID; HPRD) LCK
SQSTM1 (CCSB; GRID; HPRD) LCK STAT1 (HPRD) LCK STAT3 (CCSB; GRID;
HPRD) LCK STAT5A (HPRD) LCK SYK (CCSB; GRID; HPRD; MINT) LCK TEK
(GRID; HPRD) LCK THY1 (CCSB; GRID; HPRD) LCK TRAT1 (HPRD) LCK TRPV4
(GRID; HPRD) LCK TUB (GRID; HPRD) LCK UNC119 (CCSB; GRID; HPRD) LCK
VAV1 (HPRD) LCK WASL (IntAct) LCK ZAP70 (CCSB; GRID; HPRD; MINT)
LMTK2 CDK5 (GRID) LMTK2 CDK5R1 (GRID; HPRD) LMTK2 PPP1R2 (GRID;
HPRD) LMTK3 ZBTB16 (MDC) LTK CBL (GRID; HPRD) LTK PIK3C2B (GRID;
HPRD) LTK PLCG1 (HPRD) LTK PTPN1 (GRID; HPRD) LTK SHC1 (GRID; HPRD)
LYN ACTB (HPRD) LYN ADAM15 (MINT) LYN BANK1 (GRID; HPRD) LYN BCAR1
(GRID; HPRD) LYN CASP3 (HPRD) LYN CASP7 (HPRD) LYN CASP9 (HPRD) LYN
CBL (BIND; DIP; GRID; HPRD; MINT) LYN CBLC (GRID; HPRD) LYN CD19
(GRID; HPRD) LYN CD22 (GRID; HPRD; IntAct) LYN CD24 (GRID; HPRD)
LYN CD36 (GRID; HPRD) LYN CD79A (HPRD) LYN CD79B (HPRD) LYN CDAN3
(GRID) LYN CDC2 (GRID; HPRD) LYN CDK2 (GRID; HPRD) LYN CDK4 (GRID)
LYN CDKN1B (HPRD) LYN CRKL (HPRD) LYN CSF2RA (HPRD) LYN CSF2RB
(GRID; HPRD) LYN CSF3R (GRID; HPRD) LYN CSNK2B (BIND; GRID; HPRD;
IntAct; MINT) LYN CTLA4 (GRID; HPRD) LYN DAPP1 (HPRD) LYN DLG4
(GRID; HPRD) LYN DOK1 (HPRD) LYN DOK3 (HPRD) LYN EPOR (GRID; HPRD)
LYN EVL (GRID; HPRD) LYN FCAR (GRID; HPRD) LYN FCER1G (GRID; HPRD)
LYN FCGR2A (HPRD) LYN FCGR2B (HPRD) LYN FOLR1 (GRID; HPRD) LYN GAB2
(GRID; HPRD) LYN GAB3 (GRID; HPRD) LYN GALNAC4S-6ST (GRID; HPRD)
LYN GP6 (GRID; HPRD) LYN GRIA3 (GRID; HPRD) LYN HCLS1 (GRID; HPRD;
MINT) LYN HNRPK (HPRD) LYN IL2RB (HPRD) LYN IL7R (GRID; HPRD) LYN
INPP5D (GRID; HPRD) LYN ITPR1 (HPRD) LYN KHDRBS1 (HPRD; IntAct;
MINT) LYN LCP2 (BIND; GRID; HPRD) LYN LIME1 (HPRD) LYN MAP3K3
(IntAct; MINT) LYN MAP4K1 (HPRD) LYN MAPK3 (GRID; HPRD) LYN MATK
(HPRD) LYN MME (GRID; HPRD) LYN MS4A1 (HPRD) LYN MS4A2 (GRID; HPRD)
LYN MUC1 (GRID) LYN NEDD9 (GRID; HPRD) LYN NMT1 (GRID; HPRD) LYN
NPHS1 (GRID; HPRD) LYN PAG1 (GRID; HPRD) LYN PDE4A (GRID; HPRD) LYN
PDE4D (BIND) LYN PECAM1 (GRID; HPRD) LYN PIK3CG (GRID; HPRD) LYN
PILRB (HPRD) LYN PLAUR (DIP) LYN PLCG1 (GRID; HPRD) LYN PLCG2
(HPRD) LYN PPP1R15A (BIND; GRID; HPRD) LYN PPP1R8 (GRID; HPRD) LYN
PRAM1 (GRID; HPRD) LYN PRKCD (HPRD) LYN PRKCQ (GRID; HPRD) LYN
PRKDC (GRID; HPRD) LYN PTK2 (HPRD; MINT) LYN PTPN6 (GRID; HPRD) LYN
PTPRC (HPRD) LYN RASA1 (HPRD) LYN RGS16 (BIND; HPRD) LYN SH2B2
(HPRD) LYN SHC1 (GRID; HPRD; MINT) LYN SKAP1 (GRID; HPRD) LYN SKAP2
(GRID; HPRD) LYN SLC4A1 (HPRD) LYN SNCA (HPRD) LYN SPHK2 (IntAct)
LYN SYK (DIP; GRID; HPRD; MINT) LYN TEC (HPRD) LYN TEK (HPRD) LYN
TRAT1 (HPRD) LYN TRPV4 (GRID; HPRD) LYN TYK2 (GRID; HPRD) LYN UBB
(BIND; HPRD) LYN UNC119 (GRID; HPRD) LYN YES1 (HPRD) MATK CD36
(GRID; HPRD) MATK EWSR1 (CCSB; GRID; HPRD; IntAct) MATK NTRK1
(GRID; HPRD) MATK PXN (GRID; HPRD) MATK SRC (CCSB; GRID; HPRD)
MERTK BMPR2 (HPRD) MERTK GAS6 (GRID; HPRD; reactome) MERTK GRB2
(GRID; HPRD) MERTK LMO4 (BIND; HPRD) MERTK PROS1 (reactome) MERTK
VAV1 (HPRD) MET BAG1 (GRID; HPRD) MET CASP3 (HPRD) MET CBL (BIND;
GRID; HPRD; MINT) MET CDH1 (GRID) MET CNR1 (HPRD) MET CTNNB1 (GRID;
HPRD) MET CTTN (HPRD) MET DAPK3 (HPRD) MET DNAJA3 (HPRD) MET FAS
(HPRD) MET GAB1 (BIND; GRID; HPRD; MINT) MET GLMN (GRID; HPRD) MET
GRB2 (GRID; HPRD) MET HGF (BIND; GRID; HPRD; IntAct) MET HGFAC
(DIP) MET HGS (GRID; HPRD) MET INPP5D (BIND; HPRD) MET INPPL1
(BIND; HPRD) MET ITGB4 (HPRD) MET MUC20 (HPRD) MET PCBD2 (HPRD) MET
PIK3R1 (BIND; HPRD) MET PLCG1 (HPRD) MET PLXNB1 (HPRD; MINT) MET
POLR2A (BIND) MET PTPN11 (GRID; HPRD) MET PTPRB (HPRD) MET PTPRJ
(HPRD) MET RANBP10 (GRID; HPRD) MET RANBP9 (GRID; HPRD; MINT) MET
SH3KBP1 (GRID) MET SHC1 (GRID; HPRD) MET SMC1A (HPRD) MET SNAPIN
(HPRD) MET SNX2 (HPRD) MET SPSB1 (HPRD) MET SRC (GRID; HPRD) MET
STAT3 (GRID; HPRD) MET TAF1 (BIND) MET VAV1 (HPRD)
MST1R AKT1 (HPRD) MST1R EPOR (HPRD) MST1R GAB1 (HPRD) MST1R GRB2
(GRID; HPRD) MST1R HYAL2 (GRID; HPRD) MST1R MAX (BIND) MST1R MST1
(DIP; GRID; HPRD) MST1R MYC (BIND) MST1R PIK3R1 (GRID; HPRD) MST1R
PLCG1 (GRID; HPRD) MST1R RELA (HPRD) MST1R SFN (HPRD) MST1R SHC1
(GRID; HPRD) MST1R SRC (GRID; HPRD) MST1R YES1 (HPRD) MST1R YWHAB
(HPRD) MST1R YWHAE (HPRD) MST1R YWHAH (HPRD) MST1R YWHAQ (HPRD)
MST1R YWHAZ (HPRD) MUSK COLQ (HPRD) MUSK DOK7 (HPRD) MUSK RAPSN
(GRID; HPRD) MUSK SYNE1 (GRID; HPRD) NTRK1 ADCYAP1 (reactome) NTRK1
ADCYAP1R1 (reactome) NTRK1 ADORA2A (reactome) NTRK1 BRAF (reactome)
NTRK1 CAV1 (GRID; HPRD) NTRK1 CCNA2 (BIND) NTRK1 CPSF4 (BIND) NTRK1
CRK (HPRD; reactome) NTRK1 DNAJA3 (HPRD) NTRK1 DYNLL1 (HPRD) NTRK1
FRS2 (BIND; GRID; HPRD; reactome) NTRK1 FRS3 (BIND; GRID; HPRD)
NTRK1 GIPC1 (BIND; GRID; HPRD) NTRK1 GRB2 (GRID; HPRD) NTRK1
HIST3H3 (BIND) NTRK1 HIST4H4 (BIND) NTRK1 HRAS (reactome) NTRK1
IRS1 (HPRD) NTRK1 IRS2 (HPRD) NTRK1 KIDINS220 (HPRD; reactome)
NTRK1 KRAS (reactome) NTRK1 MAPK3 (HPRD) NTRK1 NEDD4L (HPRD) NTRK1
NGF (BIND; DIP; GRID; HPRD; reactome) NTRK1 NGFR (DIP; GRID; HPRD)
NTRK1 NRAS (reactome) NTRK1 PIK3R1 (HPRD) NTRK1 PLCG1 (GRID; HPRD;
reactome) NTRK1 PTPN1 (GRID; HPRD) NTRK1 PTPN11 (HPRD) NTRK1 RAF1
(reactome) NTRK1 RAP1A (GRID; HPRD; reactome) NTRK1 RAPGEF1
(reactome) NTRK1 RASA1 (HPRD) NTRK1 RICS (GRID; HPRD) NTRK1 RIT1
(reactome) NTRK1 RIT2 (reactome) NTRK1 RUSC1 (HPRD) NTRK1 SH2B1
(HPRD) NTRK1 SH2B2 (HPRD) NTRK1 SHC1 (BIND; HPRD; reactome) NTRK1
SHC2 (HPRD; reactome) NTRK1 SHC3 (GRID; HPRD; reactome) NTRK1
SQSTM1 (HPRD) NTRK1 STAT3 (reactome) NTRK1 UBB (HPRD) NTRK1 YWHAB
(reactome) NTRK2 ADCYAP1 (reactome) NTRK2 ADCYAP1R1 (reactome)
NTRK2 ADORA2A (reactome) NTRK2 ATP5O (BIND) NTRK2 BDNF (DIP; GRID;
HPRD) NTRK2 DOK5 (HPRD) NTRK2 DYNLL1 (GRID; HPRD) NTRK2 FRS3 (GRID;
HPRD) NTRK2 GIPC1 (GRID; HPRD) NTRK2 KIDINS220 (HPRD) NTRK2 NCK1
(GRID; HPRD) NTRK2 NCK2 (GRID; HPRD) NTRK2 NGFR (HPRD) NTRK2 NTF3
(GRID; HPRD) NTRK2 NTF4 (BIND; GRID; HPRD) NTRK2 PLCG1 (HPRD) NTRK2
PTPN1 (GRID; HPRD) NTRK2 PTPN11 (HPRD) NTRK2 SH2B1 (HPRD) NTRK2
SH2B2 (HPRD) NTRK2 SH2D1A (MINT) NTRK2 SHC1 (HPRD) NTRK2 SHC2
(HPRD) NTRK2 SHC3 (GRID; HPRD) NTRK2 SQSTM1 (GRID; HPRD) NTRK2
TNFRSF1A (BIND) NTRK2 TRAF2 (BIND) NTRK2 UBB (HPRD) NTRK3 CYP2D6
(BIND) NTRK3 DOK5 (HPRD) NTRK3 DYNLL1 (HPRD) NTRK3 FOS (MINT) NTRK3
HTR2A (HPRD) NTRK3 IRAK1 (GRID; HPRD) NTRK3 JUN (MINT) NTRK3
KIDINS220 (HPRD) NTRK3 MAPK1 (HPRD; MINT) NTRK3 MAPK3 (HPRD) NTRK3
NGFR (GRID; HPRD) NTRK3 NGFRAP1 (CCSB; GRID; HPRD) NTRK3 NTF3 (DIP;
GRID; HPRD; MINT) NTRK3 PLCG1 (GRID; HPRD) NTRK3 PMAIP1 (BIND)
NTRK3 PTPN1 (CCSB; GRID; HPRD) NTRK3 SH2D1A (MINT) NTRK3 SHC1
(CCSB; GRID; HPRD) NTRK3 SHC2 (HPRD) NTRK3 SQSTM1 (CCSB; GRID;
HPRD) NTRK3 TNFRSF1A (BIND) NTRK3 TRAF2 (BIND) PDGFRA CAV1 (CCSB;
GRID; HPRD) PDGFRA CBL (GRID) PDGFRA CRK (CCSB; GRID; HPRD) PDGFRA
CRKL (GRID; HPRD) PDGFRA GRB14 (GRID; HPRD) PDGFRA ITGAV (GRID;
HPRD) PDGFRA ITGB3 (GRID; HPRD) PDGFRA PDGFA (DIP; GRID; HPRD)
PDGFRA PDGFB (CCSB; DIP; GRID; HPRD) PDGFRA PDGFC (GRID; HPRD)
PDGFRA PDGFRB (CCSB; GRID; HPRD) PDGFRA PIK3R1 (HPRD) PDGFRA PLCG1
(GRID; HPRD) PDGFRA PTPN11 (CCSB; GRID; HPRD) PDGFRA RAPGEF1 (GRID;
HPRD) PDGFRA SHB (GRID; HPRD) PDGFRA SHF (HPRD) PDGFRA SLC9A3R1
(GRID; HPRD) PDGFRA SNX2 (GRID; HPRD) PDGFRA SNX4 (CCSB; GRID;
HPRD) PDGFRA SNX6 (GRID; HPRD) PDGFRA STAT1 (HPRD) PDGFRA STAT3
(HPRD) PDGFRA STAT5A (HPRD) PDGFRA STAT5B (HPRD) PDGFRB ARAF (HPRD)
PDGFRB BAG1 (CCSB; GRID; HPRD) PDGFRB CBL (BIND; HPRD) PDGFRB CBLC
(HPRD) PDGFRB COPA (GRID; HPRD) PDGFRB COPB1 (CCSB; GRID; HPRD)
PDGFRB CRK (BIND; CCSB; GRID; HPRD) PDGFRB EDG1 (CCSB; GRID; HPRD)
PDGFRB EIF2AK2 (GRID; HPRD) PDGFRB GAB1 (HPRD) PDGFRB GRB10 (GRID;
HPRD) PDGFRB GRB14 (GRID; HPRD) PDGFRB GRB2 (CCSB; DIP; GRID; HPRD)
PDGFRB GRB7 (HPRD) PDGFRB ITGB3 (GRID; HPRD) PDGFRB KRTAP4-12
(CCSB; GRID; HPRD; IntAct) PDGFRB NCK1 (BIND; CCSB; GRID; HPRD)
PDGFRB NCK2 (CCSB; GRID; HPRD) PDGFRB PDAP1 (CCSB; GRID; HPRD)
PDGFRB PDGFB (CCSB; DIP; GRID; HPRD) PDGFRB PDGFD (HPRD) PDGFRB
PIK3C2B (IntAct) PDGFRB PIK3CA (HPRD) PDGFRB PIK3R1 (BIND; DIP;
GRID; HPRD) PDGFRB PIK3R2 (BIND; HPRD) PDGFRB PIK3R3 (BIND; CCSB;
GRID; HPRD) PDGFRB PLAUR (BIND) PDGFRB PLCG1 (BIND; DIP; GRID;
HPRD) PDGFRB PRDX2 (HPRD) PDGFRB PTEN (MINT) PDGFRB PTK2 (CCSB;
GRID; HPRD) PDGFRB PTPN1 (HPRD) PDGFRB PTPN11 (BIND; CCSB; GRID;
HPRD) PDGFRB PTPN2 (HPRD) PDGFRB PTPRJ (HPRD) PDGFRB RAF1 (CCSB;
GRID; HPRD) PDGFRB RASA1 (BIND; CCSB; DIP; GRID; HPRD) PDGFRB
SH3KBP1 (CCSB; GRID) PDGFRB SHB (BIND) PDGFRB SHC1 (BIND; CCSB;
GRID; HPRD) PDGFRB SLC9A3R1 (GRID; HPRD; MINT) PDGFRB SLC9A3R2
(MINT) PDGFRB SNX1 (CCSB; GRID; HPRD) PDGFRB SNX2 (GRID; HPRD)
PDGFRB SNX4 (GRID; HPRD) PDGFRB SOCS1 (BIND; HPRD) PDGFRB SRC
(BIND; CCSB; GRID; HPRD) PDGFRB STAT1 (HPRD) PDGFRB STAT3 (HPRD)
PDGFRB STAT5A (DIP; HPRD) PDGFRB STAT5B (HPRD) PDGFRB SYNGAP1
(HPRD) PDGFRB TYK2 (HPRD) PDGFRB YES1 (HPRD) PTK2 ACTN1 (HPRD) PTK2
APC (GRID; HPRD) PTK2 ARHGAP26 (GRID; HPRD) PTK2 ATG12 (CCSB; GRID;
HPRD; MINT) PTK2 BCAR1 (BIND; GRID; HPRD; MINT) PTK2 BIN1 (GRID;
HPRD) PTK2 BIRC4 (reactome) PTK2 CASP3 (reactome) PTK2 CASP7
(reactome) PTK2 CCR5 (GRID; HPRD) PTK2 CD4 (CCSB; GRID) PTK2 CD47
(CCSB; GRID; HPRD) PTK2 CD79B (HPRD) PTK2 CIB1 (GRID; HPRD) PTK2
CRK (GRID; HPRD) PTK2 CRKL (GRID) PTK2 CSPG4 (GRID; HPRD) PTK2
CXCR4 (HPRD) PTK2 DCC (BIND; GRID) PTK2 DDEF1 (GRID; HPRD) PTK2
DIABLO (reactome) PTK2 DLGAP3 (HPRD) PTK2 DNM2 (BIND; HPRD) PTK2
EFS (CCSB; GRID; HPRD) PTK2 EZR (CCSB; GRID; HPRD) PTK2 GIT1 (GRID:
HPRD) PTK2 GRB2 (BIND; CCSB; GRID; HPRD) PTK2 GRB7 (CCSB; GRID;
HPRD) PTK2 GSK3B (CCSB; GRID; HPRD) PTK2 GZMB (HPRD) PTK2 HNF4A
(BIND) PTK2 IGHM (HPRD) PTK2 IRS1 (GRID; HPRD) PTK2 ITGAV (GRID;
HPRD) PTK2 ITGB1 (BIND; CCSB; GRID; HPRD) PTK2 ITGB2 (CCSB; GRID;
HPRD) PTK2 ITGB3 (BIND; GRID; HPRD) PTK2 ITGB4 (HPRD) PTK2 ITGB5
(BIND) PTK2 KCNMA1 (HPRD) PTK2 MAPK8IP3 (GRID; HPRD) PTK2 MICAL1
(GRID; HPRD) PTK2 NCAM1 (HPRD) PTK2 NCK2 (CCSB; GRID; HPRD) PTK2
NEDD8 (HPRD) PTK2 NEDD9 (GRID; HPRD) PTK2 NEO1 (BIND) PTK2 PIAS1
(HPRD) PTK2 PIK3R1 (GRID; HPRD) PTK2 PKD1 (BIND; GRID; HPRD) PTK2
PLCG1 (GRID; HPRD)
PTK2 PPP1CB (CCSB; GRID; HPRD) PTK2 PTEN (GRID) PTK2 PTPN1 (HPRD)
PTK2 PTPN11 (CCSB; GRID; HPRD) PTK2 PTPN12 (GRID; HPRD) PTK2 PTPRH
(GRID; HPRD) PTK2 PXN (BIND; GRID; HPRD; MINT) PTK2 RB1CC1 (GRID;
HPRD) PTK2 RET (HPRD) PTK2 RIPK1 (BIND; HPRD; MINT) PTK2 ROCK1
(HPRD) PTK2 SELE (GRID; HPRD) PTK2 SHC1 (BIND; CCSB; GRID; HPRD)
PTK2 SOCS2 (CCSB; GRID; HPRD; MINT) PTK2 SORBS1 (HPRD) PTK2 SRC
(BIND; CCSB; GRID; HPRD; IntAct; MINT) PTK2 STAT1 (CCSB; GRID;
HPRD) PTK2 SYK (CCSB; GRID; HPRD) PTK2 TGFB1I1 (CCSB; GRID; HPRD)
PTK2 TLN1 (GRID; HPRD) PTK2 TNFRSF1A (HPRD) PTK2 TNS1 (HPRD) PTK2
TP53 (HPRD) PTK2 TRIO (HPRD) PTK2 TRIP6 (CCSB; GRID; HPRD) PTK2 VCL
(BIND; HPRD) PTK2B ANXA6 (HPRD) PTK2B ARHGAP21 (HPRD) PTK2B ARHGAP5
(HPRD) PTK2B BCAR1 (GRID; HPRD) PTK2B CBL (GRID; HPRD) PTK2B CCR5
(GRID; HPRD) PTK2B CD2AP (GRID) PTK2B CRK (GRID; HPRD) PTK2B DCC
(GRID) PTK2B DDEF1 (HPRD) PTK2B DDEF2 (GRID; HPRD) PTK2B DLG3
(GRID; HPRD) PTK2B DLG4 (GRID; HPRD) PTK2B DLGAP3 (HPRD) PTK2B EFS
(GRID; HPRD) PTK2B EWSR1 (GRID; HPRD) PTK2B FGFR2 (HPRD) PTK2B
FGFR3 (HPRD) PTK2B FLT1 (HPRD) PTK2B FYN (GRID; HPRD) PTK2B GNA13
(GRID; HPRD) PTK2B GRB2 (GRID; HPRD) PTK2B GRIN2A (GRID; HPRD)
PTK2B GSN (GRID; HPRD) PTK2B IL7R (HPRD) PTK2B ITGB2 (GRID; HPRD)
PTK2B ITGB3 (GRID; HPRD) PTK2B JAK1 (GRID; HPRD) PTK2B JAK2 (GRID;
HPRD) PTK2B JAK3 (GRID; HPRD) PTK2B KCNA2 (GRID; HPRD) PTK2B LCK
(GRID; HPRD) PTK2B LPXN (GRID; HPRD; MINT) PTK2B LYN (GRID; HPRD)
PTK2B MAP3K4 (HPRD; MINT) PTK2B MATK (GRID; HPRD) PTK2B MCAM (GRID;
HPRD) PTK2B NEDD9 (GRID; HPRD) PTK2B NPHP1 (GRID; HPRD) PTK2B PDPK1
(HPRD) PTK2B PIK3R1 (GRID; HPRD) PTK2B PITPNM1 (GRID; HPRD) PTK2B
PITPNM2 (GRID; HPRD) PTK2B PITPNM3 (GRID; HPRD) PTK2B PRKCD (GRID;
HPRD) PTK2B PTK2 (HPRD) PTK2B PTPN11 (GRID; HPRD) PTK2B PTPN12
(GRID; HPRD) PTK2B PTPN6 (GRID; HPRD) PTK2B PXN (GRID; HPRD) PTK2B
RASA1 (GRID; HPRD) PTK2B RB1CC1 (GRID; HPRD) PTK2B SH2D3C (HPRD)
PTK2B SHC1 (GRID; HPRD) PTK2B SLC2A1 (GRID; HPRD) PTK2B SNCA (HPRD)
PTK2B SORBS1 (GRID) PTK2B SORBS2 (BIND; GRID; HPRD) PTK2B SRC
(GRID; HPRD) PTK2B STAP1 (HPRD) PTK2B STAT3 (HPRD) PTK2B SYK (GRID;
HPRD) PTK2B TGFB1I1 (BIND; GRID; HPRD) PTK2B TLN1 (GRID; HPRD)
PTK2B VAV1 (GRID; HPRD) PTK2B ZAP70 (GRID; HPRD) PTK6 IRS1 (BIND)
PTK6 IRS4 (BIND) PTK6 KHDRBS1 (GRID; HPRD) PTK6 STAP2 (GRID; HPRD)
PTK7 E2F4 (BIND) PTK7 HNF4A (BIND) RET AKAP5 (HPRD) RET CBL (HPRD;
MINT) RET CBLB (HPRD) RET CRK (HPRD) RET DOK1 (GRID; HPRD) RET DOK2
(CCSB; GRID; HPRD) RET DOK3 (HPRD) RET DOK4 (GRID; HPRD) RET DOK5
(GRID; HPRD) RET DOK6 (GRID; HPRD) RET FAU (MINT) RET FRS2 (BIND;
CCSB; GRID; HPRD) RET GAB1 (GRID; HPRD) RET GDNF (GRID; HPRD) RET
GFRA1 (CCSB; GRID; HPRD) RET GFRA4 (HPRD) RET GRB10 (GRID; HPRD)
RET GRB2 (BIND; CCSB; GRID; HPRD) RET GRB7 (CCSB; GRID; HPRD) RET
HIST2H4A (HPRD) RET JUN (BIND) RET MAPK1 (HPRD) RET MAPK14 (HPRD)
RET MAPK3 (HPRD) RET MAPK8 (HPRD) RET MAPK9 (HPRD) RET NRTN (GRID;
HPRD) RET PDLIM7 (GRID; HPRD) RET PIK3R1 (GRID; HPRD) RET PLCG1
(BIND; GRID; HPRD) RET PRKAR2A (HPRD) RET PTPN11 (HPRD) RET PTPRF
(GRID) RET SHC1 (BIND; CCSB; GRID; HPRD; MINT) RET SHC3 (BIND;
HPRD) RET SRC (CCSB; GRID; HPRD) RET STAT3 (CCSB; GRID; HPRD) ROR1
NGF (DIP) ROR2 FZD2 (HPRD) ROR2 FZD5 (HPRD) ROR2 MAGED1 (GRID;
HPRD) ROR2 WNT5A (HPRD) ROS1 LPHN1 (HPRD) ROS1 PTPN6 (GRID; HPRD)
ROS1 VAV3 (GRID; HPRD) RYK FZD8 (BIND; HPRD) RYK WNT1 (BIND; HPRD)
RYK WNT3A (BIND; HPRD) SRC ACTN1 (IntAct) SRC ADAM12 (GRID; HPRD)
SRC ADAM15 (CCSB; GRID; HPRD; MINT) SRC ADRB2 (HPRD) SRC ADRB3
(GRID; HPRD) SRC ADRBK1 (CCSB; GRID; HPRD) SRC AFAP1 (CCSB; GRID;
HPRD) SRC AFAP1L2 (HPRD) SRC AKT1 (HPRD) SRC ANKRD11 (GRID; HPRD)
SRC ANXA1 (HPRD) SRC ANXA2 (CCSB; GRID; HPRD) SRC AR (GRID) SRC
ARHGAP1 (CCSB; GRID; HPRD) SRC ARR3 (CCSB; GRID; HPRD) SRC ARRB1
(MINT) SRC ATP2B4 (HPRD) SRC BCAR1 (BIND; GRID; HPRD) SRC BCR
(BIND; HPRD) SRC CAV1 (GRID; HPRD; MINT) SRC CAV2 (HPRD) SRC CBL
(BIND; GRID; HPRD; MINT) SRC CBLC (HPRD) SRC CCNA2 (DIP) SRC CCND1
(DIP) SRC CD2AP (GRID; HPRD; MINT) SRC CD33 (HPRD) SRC CD36 (GRID;
HPRD) SRC CD46 (CCSB; GRID; HPRD) SRC CD59 (CCSB; GRID; HPRD) SRC
CDCP1 (BIND; HPRD) SRC CDH1 (GRID) SRC CDH5 (HPRD) SRC CDK5 (HPRD)
SRC CEACAM1 (HPRD) SRC CEACAM3 (HPRD) SRC CHUK (HPRD) SRC CNTNAP1
(HPRD) SRC COL1A1 (reactome) SRC COL1A2 (reactome) SRC CTNNB1
(HPRD) SRC CTNND1 (HPRD) SRC CTTN (HPRD) SRC DAB1 (HPRD) SRC DAB2
(CCSB; GRID; HPRD) SRC DAG1 (HPRD) SRC DAPP1 (HPRD) SRC DDEF1
(GRID; HPRD) SRC DGKA (HPRD) SRC DGKZ (HPRD) SRC DNM1 (HPRD; MINT)
SRC DNM2 (MINT) SRC DOK1 (HPRD) SRC DOK4 (BIND; HPRD) SRC DRD4
(BIND) SRC E2F4 (BIND) SRC EFNB1 (CCSB; GRID; HPRD) SRC EFNB2
(HPRD) SRC EGF (reactome) SRC EPS8 (CCSB; GRID; HPRD) SRC ESR1
(GRID; HPRD) SRC ESR2 (GRID; HPRD) SRC ETS1 (BIND; HPRD) SRC ETS2
(BIND; HPRD) SRC EVL (CCSB; GRID; HPRD) SRC FARP2 (HPRD) SRC FASLG
(GRID; HPRD) SRC FCER1G (reactome) SRC FHIT (HPRD) SRC FOXO1 (GRID;
HPRD) SRC FRS2 (HPRD) SRC FYB (HPRD) SRC GAB2 (GRID; HPRD) SRC GAB3
(GRID; HPRD) SRC GFAP (CCSB; GRID; HPRD) SRC GIT1 (GRID; HPRD) SRC
GJA1 (BIND; CCSB; GRID; HPRD; reactome) SRC GJB1 (BIND; HPRD) SRC
GNB2L1 (BIND; CCSB; GRID; HPRD) SRC GP2 (HPRD) SRC GP6 (reactome)
SRC GRB10 (HPRD) SRC GRB2 (CCSB; GRID; HPRD) SRC GRIN2A (GRID;
HPRD) SRC GRIN2B (GRID; HPRD) SRC GRLF1 (HPRD) SRC GTF2I (HPRD) SRC
GUCY2C (HPRD; MINT) SRC HLA-A (HPRD) SRC HLA-B (HPRD) SRC HNF1A
(GRID; HPRD) SRC HNRPK (BIND; CCSB; GRID; HPRD) SRC HRAS (HPRD;
MINT; reactome) SRC HSP90AA1 (CCSB; DIP; GRID; HPRD) SRC IKBKB
(HPRD; IntAct) SRC IKBKG (HPRD; IntAct) SRC IL6R (GRID; HPRD) SRC
INPPL1 (HPRD) SRC ITGB3 (HPRD) SRC JUP (HPRD) SRC KCNA5 (HPRD) SRC
KCNB1 (HPRD) SRC KCNQ5 (HPRD) SRC KHDRBS1 (BIND; GRID; HPRD;
IntAct; MDC; MINT) SRC KIFAP3 (CCSB; GRID; HPRD)
SRC KRAS (reactome) SRC LCT (MINT) SRC LRP1 (HPRD) SRC MAPK15
(HPRD) SRC MPZL1 (CCSB; GRID; HPRD) SRC MUC1 (BIND; GRID; HPRD;
MINT) SRC MYLK (GRID; HPRD) SRC NCOA6 (GRID) SRC ND2 (GRID; HPRD)
SRC NDUFS1 (BIND) SRC NFKBIA (HPRD) SRC NMT1 (HPRD) SRC NOS2A
(HPRD) SRC NPHS1 (GRID; HPRD) SRC NRAS (reactome) SRC P2RY2 (HPRD)
SRC PAK2 (HPRD) SRC PDE4D (BIND) SRC PDE6G (GRID; HPRD) SRC PDPK1
(HPRD) SRC PECAM1 (GRID; HPRD) SRC PELP1 (GRID; HPRD) SRC PGR
(HPRD) SRC PIK3R1 (GRID; HPRD) SRC PKD1 (GRID; HPRD) SRC PLCG1
(GRID; HPRD) SRC PLSCR1 (GRID) SRC PPARD (HPRD) SRC PRKACA (HPRD)
SRC PRKCA (HPRD) SRC PRKCD (HPRD; MINT) SRC PRKCE (GRID; HPRD) SRC
PRKCH (HPRD) SRC PRKCI (HPRD) SRC PRKCZ (CCSB; GRID; HPRD) SRC
PRKD1 (HPRD) SRC PTPN1 (HPRD) SRC PTPN11 (HPRD) SRC PTPN18 (HPRD)
SRC PTPN2 (BIND; HPRD) SRC PTPN21 (GRID; HPRD) SRC PTPN6 (HPRD) SRC
PTPRA (HPRD) SRC PTPRE (HPRD) SRC PXN (GRID; HPRD) SRC RAF1 (CCSB;
GRID; HPRD) SRC RALA (reactome) SRC RALB (reactome) SRC RALGDS
(reactome) SRC RASA1 (CCSB; GRID; HPRD) SRC RGS16 (BIND; HPRD) SRC
RICS (BIND; GRID; HPRD) SRC SH2D2A (BIND) SRC SH2D3C (HPRD) SRC
SH3BP1 (CCSB; GRID; HPRD) SRC SH3PXD2A (GRID; HPRD) SRC SHB (BIND;
GRID; HPRD) SRC SHC1 (BIND; CCSB; GRID; HPRD) SRC SKAP1 (GRID;
HPRD) SRC SLC9A2 (GRID; HPRD) SRC SMARCB1 (GRID; HPRD) SRC SMARCE1
(GRID; HPRD) SRC SNURF (GRID) SRC SPTAN1 (HPRD) SRC SRF (GRID;
HPRD) SRC STAP2 (HPRD) SRC STAT1 (CCSB; GRID: HPRD) SRC STAT3
(CCSB; GRID: HPRD) SRC STAT5A (CCSB; GRID; HPRD) SRC STAT5B (HPRD)
SRC STAT6 (HPRD) SRC SUPT4H1 (BIND) SRC SYK (CCSB; GRID; HPRD;
reactome) SRC SYN1 (GRID; HPRD) SRC TERT (HPRD) SRC TIAM1 (GRID;
HPRD) SRC TLR3 (MINT) SRC TNFRSF11A (HPRD) SRC TNFSF11 (HPRD) SRC
TRAF1 (HPRD) SRC TRAF3 (HPRD) SRC TRAF6 (CCSB; GRID; HPRD) SRC
TRAT1 (HPRD) SRC TRIP6 (CCSB; GRID; HPRD) SRC TRPC6 (GRID; HPRD)
SRC TRPV4 (GRID; HPRD) SRC TUB (GRID; HPRD) SRC TXK (HPRD) SRC
TYRO3 (GRID; HPRD) SRC VCL (HPRD) SRC VIL1 (HPRD) SRC WAS (CCSB;
GRID; HPRD) SRC WBP11 (HPRD) SRC WT1 (HPRD) SRC YTHDC1 (GRID; HPRD)
SRC YWHAB (CCSB; GRID; HPRD) SRC YWHAE (CCSB; GRID; HPRD) SRC YWHAG
(CCSB; GRID; HPRD) SRC YWHAH (GRID; HPRD) SRM MAX (BIND) SYK BLNK
(CCSB; GRID; HPRD) SYK CBL (BIND; GRID; HPRD; MINT) SYK CBLB (BIND;
CCSB; GRID; HPRD) SYK CD19 (GRID; HPRD) SYK CD22 (GRID; HPRD;
IntAct) SYK CD3E (GRID; HPRD) SYK CD79A (GRID; HPRD) SYK CD79B
(HPRD) SYK COL1A1 (reactome) SYK COL1A2 (reactome) SYK CRKL (BIND;
GRID; HPRD) SYK CSF2RB (HPRD) SYK CSF3R (GRID; HPRD) SYK CTTN
(CCSB; GRID; HPRD) SYK DBNL (HPRD) SYK DTYMK (BIND) SYK DUSP3
(HPRD) SYK EPOR (GRID; HPRD) SYK EZR (BIND; CCSB; GRID) SYK FCER1G
(GRID; HPRD; IntAct; reactome) SYK FCGR1A (CCSB; GRID; HPRD) SYK
FCGR2A (HPRD) SYK GAB2 (HPRD) SYK GP6 (reactome) SYK GRB2 (CCSB;
GRID; HPRD) SYK HCLS1 (HPRD) SYK HMGCS2 (BIND) SYK HNRNPU (GRID;
HPRD) SYK IL15RA (HPRD) SYK IL2RB (HPRD) SYK ITGB2 (HPRD; IntAct)
SYK JUN (BIND) SYK LAT (CCSB; GRID; HPRD) SYK LAX1 (HPRD) SYK LCP2
(CCSB; GRID; HPRD) SYK MAP4K1 (HPRD) SYK MAPK3 (HPRD) SYK MAX
(BIND) SYK MS4A2 (GRID; HPRD) SYK MSN (BIND) SYK MYC (BIND) SYK
NFAM1 (HPRD) SYK NFKBIB (BIND) SYK PAG1 (GRID; HPRD) SYK PIK3AP1
(HPRD) SYK PIK3R1 (HPRD; MINT) SYK PIK3R2 (HPRD; MINT) SYK PLCG1
(DIP; GRID; HPRD) SYK PLCG2 (HPRD; reactome) SYK POU2AF1 (IntAct)
SYK PRKCA (HPRD) SYK PRKD1 (HPRD) SYK PTPN6 (CCSB; GRID; HPRD) SYK
PXN (GRID; HPRD) SYK RASA1 (HPRD) SYK RPS6KA1 (HPRD) SYK RPS6KA2
(DIP) SYK SELPLG (HPRD) SYK SH2B2 (HPRD) SYK SH3BP2 (GRID; HPRD)
SYK SHC1 (HPRD) SYK SIT1 (GRID; HPRD) SYK SLA (CCSB; GRID; HPRD)
SYK SLC4A1 (HPRD) SYK SNCA (HPRD) SYK STAT1 (BIND; HPRD) SYK STAT3
(CCSB; GRID; HPRD) SYK STAT5A (CCSB; GRID; HPRD) SYK TAF1 (BIND)
SYK TLR4 (HPRD) SYK TRAF6 (CCSB; GRID; HPRD) SYK TUBA1A (CCSB;
GRID; HPRD) SYK TUBA4A (HPRD) SYK TYROBP (HPRD) SYK UBB (BIND;
CCSB; GRID; HPRD) SYK UCP2 (BIND) SYK VAV1 (CCSB; GRID; HPRD) SYK
VAV2 (HPRD) TEC DOK1 (HPRD) TEC EPOR (HPRD) TEC GNA12 (GRID; HPRD)
TEC IL3RA (HPRD) TEC PIK3R1 (HPRD) TEC PIK3R3 (GRID; HPRD) TEC
PIP4K2A (HPRD) TEC PIP5K1A (HPRD) TEC PLCG1 (HPRD) TEC PLCG2 (HPRD)
TEC PLK4 (GRID; HPRD) TEC PRLR (GRID; HPRD) TEC PTPN18 (HPRD) TEC
PTPN21 (GRID; HPRD) TEC SHC1 (HPRD) TEC SOCS1 (GRID; HPRD) TEC
STAP1 (HPRD) TEC VAV1 (GRID; HPRD) TEC WAS (GRID; HPRD) TEK ANGPT1
(DIP; GRID; HPRD) TEK ANGPT2 (BIND; DIP; GRID; HPRD) TEK ANGPT4
(BIND; GRID; HPRD) TEK ANGPTL1 (BIND; GRID; HPRD) TEK DOK2 (GRID;
HPRD) TEK DOK4 (HPRD) TEK GRB14 (GRID; HPRD) TEK GRB2 (GRID; HPRD)
TEK GRB7 (GRID; HPRD) TEK PIK3R1 (GRID; HPRD) TEK PTPN11 (GRID;
HPRD) TEK PTPRB (HPRD) TEK SHC1 (HPRD) TEK SOCS1 (BIND; GRID; HPRD)
TEK STAT5A (HPRD) TEK STAT5B (HPRD) TEK TIE1 (GRID; HPRD) TEK TNIP2
(HPRD) TIE1 PIK3R1 (GRID; HPRD) TIE1 PTPN11 (CCSB; GRID; HPRD) TNK1
PLCG1 (GRID; HPRD) TNK1 SFN (GRID) TNK2 ARSE (HPRD; MDC) TNK2 CDC42
(CCSB; GRID; HPRD) TNK2 CLTC (GRID; HPRD) TNK2 CSPG4 (GRID; HPRD)
TNK2 GRB2 (CCSB; GRID; HPRD) TNK2 HSH2D (GRID; HPRD) TNK2 HSP90AB2P
(HPRD) TNK2 ITFG2 (HPRD; MDC) TNK2 MCF2 (GRID; HPRD) TNK2 MERTK
(HPRD) TNK2 NCK1 (CCSB; GRID; HPRD) TNK2 RASGEF1C (HPRD; MDC) TNK2
RASGRF1 (GRID; HPRD) TNK2 RPL18A (HPRD; MDC) TNK2 SEZ6 (HPRD; MDC)
TNK2 SFRS5 (HPRD; MDC) TNK2 SNX9 (HPRD) TNK2 VTI1B (HPRD; MDC) TNK2
WWOX (HPRD) TXK CCR5 (GRID; HPRD) TXK CTLA4 (HPRD) TXK LCP2 (HPRD)
TXK SH2D2A (HPRD) TXK TH1L (HPRD) TXK YTHDC1 (GRID; HPRD) TYK2 CBL
(BIND; GRID; HPRD) TYK2 CRKL (GRID; HPRD) TYK2 GHR (GRID; HPRD)
TYK2 GNB2L1 (BIND; CCSB; GRID; HPRD; MINT) TYK2 IFNAR1 (BIND; CCSB;
DIP; GRID; HPRD; MINT) TYK2 IFNAR2 (BIND; HPRD) TYK2 IL12RB1
(BIND)
TYK2 IL13RA1 (CCSB; GRID; HPRD) TYK2 IL6ST (DIP; GRID; HPRD) TYK2
IRS1 (GRID; HPRD) TYK2 IRS2 (GRID; HPRD) TYK2 JAKMIP1 (BIND; HPRD)
TYK2 MAX (BIND) TYK2 MYC (BIND) TYK2 PIK3R1 (GRID; HPRD) TYK2 PLAUR
(BIND; DIP; HPRD) TYK2 PRMT5 (GRID; HPRD) TYK2 PTAFR (HPRD) TYK2
PTPN1 (CCSB; GRID; HPRD) TYK2 PTPN6 (CCSB; GRID; HPRD) TYK2 PTPRC
(HPRD) TYK2 STAM2 (CCSB; GRID; HPRD) TYK2 STAT1 (GRID; HPRD; MINT)
TYK2 STAT2 (HPRD) TYK2 TAF1 (BIND) TYK2 VAV1 (CCSB; GRID; HPRD)
TYK2 XRCC5 (GRID; HPRD) TYRO3 GAS6 (GRID; HPRD) TYRO3 PIK3R1 (GRID;
HPRD) TYRO3 PROS1 (GRID; HPRD) TYRO3 YES1 (GRID; HPRD) YES1 ADAM12
(GRID; HPRD) YES1 ADAM15 (MINT) YES1 CD2AP (GRID; HPRD; MINT) YES1
CD36 (GRID; HPRD) YES1 CD46 (GRID; HPRD) YES1 CDCP1 (BIND) YES1
CKAP4 (IntAct) YES1 DLG4 (GRID; HPRD) YES1 DOK1 (HPRD) YES1 GP2
(HPRD) YES1 GP6 (HPRD) YES1 ITGB4 (HPRD) YES1 NPHS1 (GRID; HPRD)
YES1 OCLN (GRID; HPRD) YES1 PECAM1 (GRID; HPRD) YES1 PTPRE (HPRD)
YES1 RASA1 (GRID; HPRD) YES1 RPL10 (GRID; HPRD) YES1 TAF1 (BIND)
YES1 TP53BP2 (GRID; HPRD) YES1 TRPV4 (GRID; HPRD) YES1 YAP1 (GRID)
YES1 ZNF512B (BIND; MINT) ZAP70 ACP1 (CCSB; GRID; HPRD) ZAP70
CARD11 (HPRD) ZAP70 CBL (GRID; HPRD) ZAP70 CBLB (CCSB; GRID; HPRD)
ZAP70 CD247 (BIND; CCSB; GRID; HPRD) ZAP70 CD3E (GRID; HPRD) ZAP70
CD5 (GRID; HPRD) ZAP70 CD79B (HPRD) ZAP70 CRK (BIND; CCSB; GRID;
HPRD) ZAP70 CRKL (BIND) ZAP70 DBNL (CCSB; GRID; HPRD) ZAP70 DEF6
(HPRD) ZAP70 DUSP3 (HPRD) ZAP70 FCGR3A (CCSB; GRID; HPRD) ZAP70
FCRL3 (GRID; HPRD) ZAP70 GAB2 (GRID; HPRD) ZAP70 GRB2 (HPRD) ZAP70
HSP90AA1 (MINT) ZAP70 IFNAR1 (HPRD) ZAP70 LAT (HPRD; MINT) ZAP70
LAX1 (HPRD) ZAP70 LCP2 (HPRD) ZAP70 MUC1 (HPRD) ZAP70 NFAM1 (HPRD)
ZAP70 PAG1 (GRID; HPRD) ZAP70 PLCG1 (GRID; HPRD) ZAP70 PRLR (GRID;
HPRD) ZAP70 PTPN3 (HPRD) ZAP70 PTPN6 (CCSB; GRID; HPRD) ZAP70 PTPRC
(GRID; HPRD) ZAP70 RASA1 (CCSB; GRID; HPRD) ZAP70 SH2B3 (HPRD)
ZAP70 SH3BP2 (GRID; HPRD) ZAP70 SHB (GRID; HPRD) ZAP70 SHC1 (BIND;
CCSB; GRID; HPRD) ZAP70 SIT1 (GRID; HPRD) ZAP70 SLA (CCSB; GRID;
HPRD) ZAP70 SLA2 (GRID; HPRD) ZAP70 SLAMF6 (GRID; HPRD) ZAP70 SOS1
(GRID; HPRD) ZAP70 TUBA4A (CCSB; GRID; HPRD) ZAP70 TUBB (HPRD)
ZAP70 TUBB2A (HPRD) ZAP70 TYROBP (HPRD) ZAP70 UBE2L3 (HPRD) ZAP70
VAV1 (CCSB; GRID; HPRD) ZAP70 WIPF1 (HPRD)
[0242] A list of the interacting proteins of Table 5 followed by
their accession numbers (in parenthesis) are as follows:
[0243] AATK (205986_at), ABI1 (209027_s_at 209028_s_at), ABI2
(225098_at 209856_x_at 207268_x_at 225112_at 211793_s_at), ABL1
(202123_s_at), ABL2 (231907_at 206411_s_at 226893_at), ACP1
(1554808_at 201630_s_at 215227_x_at 201629_s_at), ACPP (237030_at
231711_at 204393_s_at), ACTA1 (203872_at), ACTB
(AFFX-HSAC07/X00351_3_at 213867_x_at AFFX-HSAC07/X00351_M_at
AFFX-HSAC07/X00351_5_at 224594_x_at 200801_x_at), ACTN1
(208637_x_at 208636_at), ADAM10 (202604_x_at 233538_s_at
214895_s_at), ADAM12 (202952_s_at 213790_at 204943_at), ADAM15
(217007_s_at 1555896_a_at), ADAM17 (205746_s_at 205745_x_at),
ADCYAP1 (230237_at 206281_at), ADCYAP1R1 (207151_at 236373_at
226690_at 242547_at), ADD2 (205268_s_at 237336_at 206807_s_at),
ADH6 (214261_s_at 207544_s_at), ADORA2A (205013_s_at), ADRB2
(206170_at), ADRB3 (206812_at 217303_s_at), ADRBK1 (201401_s_at),
AFAP1 (203563_at), AFAP1L2 (226829_at), AGK (1555610_at 222132_s_at
218568_at), AGTR1 (205357_s_at 208016_s_at), AGTR2 (207293_s_at
222321_at 207294_at), AHSG (210929_s_at 204551_s_at), AKAP5
(230846_at 207800_at), AKT1 (207163_s_at), ALCAM (1569362_at
240655_at 201951_at 201952_at), ALK (208211 s_at 208212 s_at), AMH
(206516 at), ANGPT1 (241119 at 1552939 at 205609 at 205608 s_at),
ANGPT2 (237261 at 205572 at 236034 at 211148 s_at), ANGPT4
(221134_at), ANGPTL1 (239183_at 231773_at 224339_s_at), ANKRD11
(238538_at 234701_at 227661_at 219437_s_at 228356_at 231999_at),
ANKS1A (212747_at), ANXA1 (201012_at), ANXA2 (210427_x_at
208816_x_at 213503_x_at 201590_x_at 1568126_at), ANXA5 (200782_at),
ANXA6 (200982_s_at), AP2A1 (234068_s_at 223237_x_at 229617_x_at),
AP2A2 (212159_x_at 211779_x_at 215764_x_at 212161_at), AP2B1
(200615_s_at 200612_s_at), AP2M1 (200613_at), AP2S1 (202120_x_at
211047_x_at 208074_s_at), APBA3 (205146_x_at 215148_s_at), APBB1
(202652_at), APBB2 (213419_at 212972_x_at 212985_at 216747_at
216750_at 40148_at 212970_at), APBB3 (204650_s_at), APC (215310_at
203526_s_at 203525_s_at 203527_s_at 216933_x_at), APP (200602_at
211277_x_at 237571_at 214953_s_at 243314_at), APPL1 (222538_s_at
218158_s_at), AQP1 (209047_at 207542_s_at), AR (211621_at
211110_s_at), ARAF (201895_at), AREG (205239_at), ARF1 (208750_s_at
200065_s_at 244504_x_at), ARF4 (201096_s_at 201097_s_at), ARHGAP1
(216689_x_at 241419_at 217153_at 202117_at), ARHGAP21 (241701_at
224764_at), ARHGAP26 (226576_at 205069_s_at 205068_s_at
215955_x_at), ARHGAPS (233849_s_at 217936_at 235635_at
1552627_a_at), ARHGEF12 (237398_at 234541_s_at 233620_at
233621_s_at 201335_s_at 234129.sub.-at 201333_s_at 201334_s_at),
ARHGEF15 (217348_x_at 205507_at), ARHGEF6 (209539_at), ARID3A
(205865_at), ARR3 (207136_at), ARRB1 (228444_at 222912_at 49111_at
222756_s_at 43511_s_at 218832_x_at 221861_at), ARSE (205894_at),
ATF2 (1555146_at 205446_s_at), ATF3 (1554420_at 1554980_a_at
202672_s_at), ATF7IP (231825_x_at 218987_at 216197_at 216198_at),
ATG12 (213930_at 213026_at 204833_at), ATIC (208758_at), ATM
(1554631_at 210858_x_at 208442_s_at 212672_at 1570352_at
1553387_at), ATP1A1 (220948_s_at), ATP1B1 (227556_at 201243_s_at
201242_s_at), ATP2B4 (212136_at 205410_s_at 212135_s_at), ATP5C1
(205711_x_at 208870_x_at 213366_x_at), ATPSH (210149_s_at
1555998_at), ATP50 (1564482_at 200818_at), ATP6V1H (1557585_at
1557586_s_at 221504_s_at), ATR (209902_at 233288_at 209903_s_at),
ATXN1 (236404_at 1559249_at 203232_s_at 203231_s_at 242230_at
230507_at 236802_at), AXL (202685_s_at 202686_s_at), BAG1
(202387_at 229720_at 211475_s_at), BANK1 (1558662_s_at 222915_s_at
219667_s_at), BCAR1 (223116_at), BCAS2 (203053_at), BCL2 (203685_at
207005_s_at 207004_at 203684_s_at 237837_at), BCL2L1 (215037_s_at
212312_at 206665_s_at), BCL3 (204908_s_at 204907_s_at), BCR
(226602_s_at 202315_s_at 217223_s_at), BDNF (239367_at 244503_at
206382_s_at), BIN1 (214643_x_at 202931_x_at 214439_x_at 210202_s_at
210201_x_at), XIAP (225858_s_at 235222_x_at 206536_s_at 225859_at
243026_x_at 228363_at), BLK (244394_at 236820_at 206255_at
210934_at), BLNK (244172_at 207655_s_at 243867_at), BMPR2
(238516_at 225144_at 210214_s_at 209920_at 231873_at), BMX
(206464_at 242967_at), BNIP2 (209308_s_at), BRAF (206044_s_at
243829_at), BRCA1 (211851_x_at 204531_s_at), BTC (241412_at
207326_at), BTK (205504_at), C13orf34 (219544_at), C14orf4 (not
found), BENDS (219670_at), C2orf44 (219120_at), C3 (217767_at),
C6orf47 (204968_at), CABLES1 (225531_at 225532_at), CABLES2
(226004_at), CACNA1D (207998_s_at 210108_at), CALM1 (213710_s_at
213688_at 211985_s_at 211984_at 209563_x_at 200653_s_at
200655_s_at), CALM2 (207243_s_at), CALM3 (1563431_x_at 200622_x_at
200623_s_at), CAMK2A (213108_at 207613_s_at), CAMK2G (212669_at
212757_s_at), CAMLG (203538_at), CARD11 (223514_at), CASP1
(211366_x_at 209970_x_at 211368_s_at 211367_s_at 1552703_s_at),
CASP3 (202763_at), CASP7 (207181_s_at), CASP8 (207686_s_at
1553306_at 213373_s_at), CASP9 (237451_x_at 240437_at 203984_s_at
210775_x_at), CAT (201432_at 211922_s_at), CAV1 (212097_at
203065_s_at), CAV2 (213426_s_at 203323_at 203324_s_at), CAV3
(208204_s_at), CBL (225231_at 229010_at 206607_at 243475_at
225234_at), CBLB (208348_s_at 209682_at 227900_at), CBLC
(220638_s_at 223668_at), CCDC17 (236320_at), CCNA2 (213226_at
203418_at), CCND1 (208712_at 208711_s_at), CCR1 (205099_s_at
205098_at), CCR2 (207794_at 206978_at), CCR3 (208304_at), CCRS
(206991_s_at), CD19 (206398_s_at), CD2 (205831_at), CD22 (220674_at
204581_at 38521_at 217422_s_at), CD226 (207315_at), CD24
(216379_x_at 208651_x_at 209771_x_at 266_s_at 209772_s_at
208650_s_at), CD247 (210031_at), CD28 (206545_at 211856_x_at
211861_x_at), CD2AP (203593_at 236257_at), CD33 (206120_at), CD36
(228766_at 209554_at 206488_s_at 209555_s_at), CD38 (234187_at
205692_s_at), CD3E (205456_at), CD4 (216424_at 203547_at), CD40
(205153_s_at 35150_at 222292_at 215346_at), CD44 (1565868_at
212014_x_at 229221_at 212063_at 209835_x_at 217523_at 1557905_s_at
210916_s_at 204490_s_at 216056_at 204489_s_at), CD46 (208783_s_at
211574_s_at 207549_x_at), CD47 (213856_at 213055_at 213857_s_at
226016_at 211075_s_at 242974_at 227259_at), CD48 (237759_at
204118_at), CD5 (230489_at 206485_at), CD55 (1555950_a_at
201925_s_at 201926_s_at), CD59 (228748_at 200983_x_at 212463_at
200984_s_at 200985_s_at), CD63 (200663_at), CD79A (205049_s_at
1555779_a_at), CD79B (205297_s_at 1555748_x_at 1555746_at), CD81
(200675_at), CD82 (228910_at 203904_x_at), CD8A (205758_at), CD8B
(230037_at 215332_s_at 207979_s_at), CD9 (201005_at), CDAN3 (not
found), CDK1 (231534_at 203214_x_at 210559_s_at 203213_at), CDC25A
(1555772_a_at 204696_s_at 204695_at), CDC25C (217010_s_at
205167_s_at), CDC37 (209953_s_at), CDC42 (210232_at 214230_at
208728_s_at 208727_s_at), CDCP1 (218451_at 1554110_at 234932_s_at),
CDH1 (201131_s_at 201130_s_at), CDH2 (203440_at 203441_s_at), CDHS
(204677_at), CDK2 (211804_s_at 204252_at 211803_at), CDK4
(202246_s_at), CDKS (204247_s_at), CDK5R1 (204996_s_at 204995_at),
CDKN1B (209112_at), CEACAM1 (211889_x_at 209498_at 206576_s_at
210610_at 211883_x_at), CEACAM3 (208052_x_at 210789_x_at), CEBPB
(212501_at), CENPF (207828_s_at 209172_s_at), ARAP1 (34206_at
212516_at), CHGB (204260_at), CHN1 (212624_s_at), CHN2 (207486_x_at
213385_at 211419_s_at), CHUK (209666_s_at), CIB1 (201953_at), CISH
(221223_x_at 223377_x_at 223961_s_at), CKAP4 (200998_s_at
200999_s_at 226526_s_at), CLDN4 (201428_at), CLTA (1560434_x_at
200960_x_at 1560433_at 216295_s_at 204050_s_at), CLTC (200614_at),
CLTCL1 (205944_s_at), CMTM3 (1555704_at 1555705_a_at 224733_at),
CNN1 (203951_at), CNN3 (201445_at), CNR1 (213436_at 208243_s_at
207940_x_at), CNTN1 (241190_at 211203_s_at 227202_at), CNTNAP1
(219400_at), COL11A1 (229271_x_at 37892_at 204320_at), COL18A1
(209082_s_at 209081_s_at), COL1A1 (1556499_s_at 202310_s_at
202312_s_at 217430_x_at 202311_s_at), COL1A2 (202404_s_at
202403_s_at), COL2A1 (217404_s_at 213492_at), COL3A1 (211161_s_at
215076_s_at 201852_x_at), COL5A2 (221729_at 221730_at), COLO
(206073_at), COPA (214336_s_at 1559862_at 208684_at), COPB1
(201359_at 201358_s_at), PTGS2 (204748_at 1554997_a_at), COX6C
(201754_at), COX7B (202110_at), CPSF4 (206688_s_at), CREB1
(204313_s_at 225565_at 214513_s_at 204314_s_at 237289_at 243625_at
204312_x_at 225572_at), CREBBP (211808_s_at 202160_at 228177_at
237239_at), CRK (202224_at 202226_s_at), CRKL (206184_at
212180_at), CRLF2 (208303_s_at), CSF1 (211839_s_at 210557_x_at
207082_at 209716_at), CSF1R (203104_at), CSF2RA (210340_s_at
211287_x_at 211286_x_at 207085_x_at), CSF2RB (205159_at), CSF3R
(203591_s_at 1553297_a_at), CSK (202329_at), CSNK2B (201390_s_at),
CSPG4 (214297_at 204736_s_at), CTGF (209101_at), CTLA4 (221331_x_at
231794_at 234895_at 236341_at 234362_s_at), CTNNB1 (1554411_at
201533_at), CTNND1 (211240_x_at 208862_s_at 208407_s_at
1557944_s_at), CTNND2 (209617_s_at 209618_at), CTR9 (202060_at),
CTSK (202450_s_at), CTTN (201059_at 214782_at 214074_s_at), CXCR4
(211919_s_at 217028_at 209201_x_at), CYP2D6 (207498_s_at 217468_at
215809_at), DAB1 (228329_at 220611_at), DAB2 (210757_x_at 201278_at
232898_at 201279_s_at 201280_s_at), DAG1 (212128_s_at 205417_s_at),
DAPK3 (203891_s_at 228681_x_at 203890_s_at), DAPP1 (222859_s_at
222858_s_at 219290_x_at), DBNL (222429_at), DCC (238914_at
206939_at), DCN (239786_at 201893_x_at 211896_s_at 211813_x_at
209335_at 229554_at), DDB1 (208619_at), DDB2 (203409_at), ASAP1
(231205_at 237082_at 224790_at 224791_at 236533_at 221039_s_at
224796_at), ASAP2 (206414_s_at), DDR1 (208779_x_at 207169_x_at
210749_x_at 1007 s_at), DDR2 (227561_at 205168_at), DEF6
(221293_s_at 226659_at), DEGS1 (207431_s_at 209250_at), DGKA
(211272_s_at 203385_at), DGKZ (207556_s_at 239342_at), DIABLO
(219350_s_at), DLG1 (229703_at 202515_at 215988_s_at 217208_s_at
202516_s_at 202514_at), DLG2 (228973_at 206253_at), DLG3 (241839_at
207732_s_at 212729_at 212727_at 212728_at), DLG4 (204592_at
210684_s_at), DLGAP3 (231151_at), DNAJA3 (205963_s_at
1554078_s_at), DNM1 (215116_s_at 217341_at), DNM2 (202253_s_at
216024_at), DOK1 (216835_s_at 211121_s_at), DOK2 (214054_at), DOK3
(223553_s_at 220320_at), DOK4 (207747_s_at 209690_s_at
209691_s_at), DOKS (214844_s_at 1554863_s_at), DOK6 (236290_at
242867_x_at 241729_at 231980_at), DOK7 (240633_at), DPM2
(209391_at), DPYSLS (224100_s_at 222797_at), DRD4 (208215_x_at),
DTYMK (1553984_s_at 203270_at), DUSP3 (201537_s_at 201536_at
201538_s_at), DYNLL1 (200703_at), E2F4 (38707_r at 202248_at),
S1PR1 (239401_at 204642_at 244422_at), EFNA1 (202023_at), EFNA2
(208256_at 1553573_s_at), EFNA3 (210132_at), EFNA4 (205107_s_at),
EFNAS (227955_s_at 214036_at 207301_at 233814_at), EFNB1
(202711_at), EFNB2 (202669_s_at 202668_at), EFNB3 (210883_x_at
205031_at), EFS (210880_s_at 204400_at), EGF (206254_at), EGFR
(211550_at 210984_x_at 211551_at 211607_x_at 201984_s_at
201983_s_at 1565483_at 1565484_x_at), EGR1 (201694_s_at 201693_s_at
227404_s_at), EHD1 (222221_x_at 209039_x_at 209038_s_at 209037_s_at
208112_x_at), EIF2AK2 (204211_x_at), EIF3E (236989_at 208697_s_at
235429_at), EIF4B (211938_at 219599_at 211937_at), ELF3 (229842_at
201510_at 210827_s_at), ELMO1 (204513_s_at), ELP2 (231713_s_at
232503_at), ENPP1 (228952_at 229088_at 205066_s_at 205065_at),
EP300 (213579_s_at 202221_s_at), EPB41 (207793_s_at 214530_x_at
225051_at 1554481_a_at), EPHA1 (215804_at 205977_s_at), EPHA2
(203499_at), EPHA3 (206071_s_at 206070_s_at 211164_at), EPHA4
(227449_at 228948_at 229374_at 206114_at), EPHAS (215664_s_at
216837_at 237939_at), EPHA6 (233184_at 1561396_at), EPHA7
(1554629_at 229288_at 238533_at 206852_at), EPHA8 (231796_at
1554069_at), EPHB1 (230425_at 211898_s_at 210753_s_at), EPHB2
(209588_at 209589_s_at 211165_x_at 210651_s_at), EPHB3 (204600_at
1438_at), EPHB4 (202894_at 216680_s_at), EPHB6 (204718_at), EPOR
(209962_at 216999_at 209963_s_at 396_f at 3798_at 215054_at), EPPK1
(232164_s_at 208156_x_at 232165_at), EPS15 (217886_at 234277_at
217887_s_at 234278_at), EPS8 (202609_at), ERBB2 (210930_s_at
216836_s_at), ERBB2IP (217941_s_at 222473_s_at), ERBB3 (1563252_at
202454_s_at 226213_at 1563253_s_at), ERBB4 (206794_at 241581_at
233494_at 214053_at 233498_at), EREG (1569583_at 205767_at), ERF
(203643_at), ERRFI1 (224657_at), ESR1 (217163_at 211627_x_at
205225_at 215552_s_at 211233_x_at 211235_s_at 211234_x_at), ESR2
(211120_x_at 211119_at 240777_at 211117_x_at 211118_x_at
210780_at), ETS1 (224833_at 1555355_a_at 241435_at 214447_at), ETS2
(242784_at 201329_s_at 201328_at), EVL (244375_at 217838_s_at
227232_at), EWSR1 (217622_at 210011_s_at 210012_s_at 229966_at
209214_s_at), EZR (217234_s_at 208622_s_at 208621_s_at 217230_at
208623_s_at), FABP4 (203980_at), FARP2 (1558855_at 1554337_at
204511_at), FAS (216252_x_at 215719_x_at 204780_s_at 204781_s_at),
FASLG (211333_s_at 210865_at), FAU (200019_s_at), FBP1 (209696_at),
FCAR (211306_s_at 211816_x_at 211305_x_at 207674_at 211307_s_at),
FCER1G (204232_at 1554899_s_at), FCER2 (206759_at 206760_s_at),
FCGR1A (214511_x_at 216950_s_at 216951_at), FCGR2A (211395_x_at
210992_x_at 203561_at 1565674_at 1565673_at), FCGR2B (211395_x_at
210889_s_at), FCGR3A (204007_at 204006_s_at), FCRL3 (231093_at
1553196_a_at), FDPS (201275_at), FER (227579_at 206412_at), FES
(205418_at), FGA (205650_s_at 205649_s_at), FGF1 (205117_at
208240_s_at 1552721_a_at), FGF10 (231762_at), FGF16 (221374_at),
FGF17 (221376_at), FGF18 (231382_at 211485_s_at 206987_x_at
211029_x_at), FGF19 (223761_at), FGF2 (204422_s_at 204421_s_at),
FGF20 (220394_at), FGF22 (221315_s_at), FGF23 (221166_at), FGF3
(214571_at), FGF4 (1552982_a_at), FGF5 (210310_s_at 210311_at
208378_x_at), FGF6 (208417_at), FGF7 (1555103_s_at 205782_at
1555102_at 1554741_s_at), FGF8 (208449_s_at), FGF9 (239178_at
206404_at), FGFR1 (215404_x_at 207822_at 226705_at 211535_s_at
210973_s_at 207937_x_at), FGFR2 (211399_at 203639_s_at 208225_at
208234_x_at 211398_at 211401_s_at 208228_s_at 203638_s_at
211400_at), FGFR3 (204380_s_at 204379_s_at), FGFR4 (1554961_at
211237_s_at 204579_at 1554962_a_at), FGR (208438_s_at), FHIT
(206492_at), FIGF (206742_at), FIZ1 (228663_x_at 226967_at), FLOT1
(210142_x_at 208749_x_at 208748_s_at), FLOT2 (201350_at
211299_s_at), FLT1 (210287_s_at 226497_s_at 222033_s_at 226498_at),
FLT3 (206674_at), FLT3LG (206980_s_at 210607_at), FLT4 (234379_at
210316_at 229902_at), FLYWCH1 (223950_s_at 234106_s_at 225952_at),
FN1 (211719_x_at 235629_at 214702_at 214701_s_at 212464_s_at
1558199_at 216442_x_at 210495_x_at), FNBP4 (235101_at 212232_at),
FOLR1 (204437_s_at), FOS (209189_at), FOXO1 (228484_s_at 239728_at
202724_s_at 202723_s_at), FRK (207178_s_at), FRS2 (226045_at
221308_at 238486_at), FRS3 (219907_at), FYB (224148_at 211795_s_at
205285_s_at 211794_at 227266_s_at), FYN (217697_at 210105_s_at
216033_s_at), FZD2 (210220_at 238129_s_at), FZD5 (221245_s_at
206136_at), FZD8 (216587_s_at 224325_at 227405_s_at), GAB1
(225998_at 214987_at 229114_at 1560382_at 226002_at 207112_s_at),
GAB2 (203853_s_at), GAB3 (241259_at 228410_at), CHST15 (244874_at
203066_at), GAPDH (AFFX-HUMGAPDH/M33197_3_at
AFFX-HUMGAPDH/M33197_5_at AFFX-HUMGAPDH/M33197_M at 212581_x_at
217398_x_at 213453_x_at), GAS6 (202177_at 1598_g_at), GDNF
(221359_at 230090_at), GFAP (203539_s_at 229259_at 203540_at),
GFRA1 (205696_s_at 227550_at 230163_at), GFRA4 (234868_s_at 221199
_at), GHR (241584_at 205498_at), GIGYF1 (228755_at 226768_at
236447_at), GIPC1 (207525_s_at), GIT1 (218030_at), GJA1
(201667_at), GJB1 (204973_at), GJC1 (228563_at 208460_at 228776_at
243502_at), GLMN (207153_s_at), GLRX (206662_at 20927_s_at), GNA11
(214679_x_at 40562_at 204248_at 564_at 213944_x_at 213766_x_at
221955_at), GNA12 (221737_at 224681_at), GNA13 (224761_at 227539_at
206917_at), GNAI1 (209576_at 227692_at), GNAI2 (201040_at), GNAQ
(211426_x_at), GNB2L1 (200651_at), GNG2 (223943_s_at 224965_at
224964_s_at 1555766_a_at), GP2 (208473_s_at 214324_at 206681_x_at),
GP6 (220336_s_at), GPSM3 (204265_s_at 214847_s_at), GPT
(206709_x_at), GPX1 (200736_s_at), GRAP (229726_at 206620_at),
GRAP2 (208406_s_at), GRB10 (209410_s_at 215248_at 209409_at
210999_s_at), GRB14 (206204_at), GRB2 (223049_at 215075_s_at), GRB7
(210761_s_at), GRIA3 (1569290_s_at 208032_s_at 206730_at
230144_at), GRIN1 (205914_s_at 205915_x_at 211125_x_at 210781_x_at
210782_x_at), GRIN2A (231384_at 206534_at 242286_at), GRIN2B
(237933_at 210411_s_at 210412_at), GRIN2D (229883_at 207036_x_at),
GRLF1 (202044_at 229394_s_at 202045_s_at 239456_at 202046_s_at),
GSK3B (242336_at 209945_s_at 226191_at), GSN (214040_s_at
200696_s_at 234240_at 227957_at 234431_at 227958_s_at), GSTO1
(1557915_s_at 201470_at), GTF2I (210891_s_at 239649_at 229896_at
210892_s_at 201065_s_at), GTF3C1 (202320_at 35671_at), GUCY2C
(206312_at), GYS1 (201673_s_at), GZMB (210164_at), H3F3A
(200080_s_at 213828_x_at 208755_x_at 213826_s_at 211940_x_at),
HBEGF (38037_at 203821_at 222076_at), HBZ (206647_at), HCK
(208018_s_at), HCLS1 (202957_at), HDHD2 (223155_at), HES1
(203395_s_at 203394_s_at 237115_at), HESS (239230_at), HGF
(210997_at 209960_at 210998_s_at 210755_at 209961_s_at), HGFAC
(207027_at), HGS (232627_at 210428_s_at), HIST2H4A (207046_at
230795_at), HIST3H3 (208572_at), HIST1H4I (214634_at), HLA-A
(221875_x_at 217436_x_at 215313_x_at 211911_x_at 204806_x_at
208729_x_at), HLA-B (216526_x_at 211911_x_at 214459_x_at
209140_x_at
208729_x_at), HMGA1 (206074_s_at 210457_x_at), HMGCS2 (204607_at
240110_at), HNF1A (210515_at), HNF4A (214832_at 230914_at
208429_x_at 216889_s_at), HNRNPC (214737_x_at 200751_s_at
200014_s_at 212626_x_at 227110_at), HNRNPL (35201_at 202072_at),
HNRNPU (225805_at 200594_x_at 200593_s_at 235603_at 216855_s_at),
HNRNPA3P1 (211931_s_at 206809_s_at 211933_s_at 206808_at), HNRNPK
(200097_s_at 200775_s_at), HOXC10 (218959_at), HRAS (212983_at),
HSH2D (1552623_at), HSP9OAA1 (214328_s_at 211969_at 210211_s_at
211968_s_at), HSP90AB2P (1557910_at 214359_s_at), HSP90B1
(216449_x_at 200598_s_at 200599_s_at), HSPA8 (210338_s_at
221891_x_at 224187_x_at 208687_x_at), HSPD1 (200806_s_at
200807_s_at), HTR2A (211616_s_at 244130_at 207135_at), HTR6
(1552857_a_at), HTT (202389_s_at 202390_s_at), HYAL2 (206855_s_at),
IBTK (210970_s_at), ICAM1 (202637_s_at 215485_s_at 202638_s_at),
IFNAR1 (225661_at 225669_at 204191_at), IFNAR2 (204785_x_at
230735_at 204786_s_at), IFNGR1 (202727_s_at 242903_at 211676_s_at),
IFNGR2 (201642_at), IGF1 (211577_s_at 209542_x_at 209541_at
209540_at), IGF1R (208441_at 243358_at 203627_at 225330_at
238544_at 237377_at 237881_at 203628_at), IGF2 (210881_s_at
202410_x_at 202409_at), IGFBP3 (210095_s_at 212143_s_at), IGHM
(212827_at), IKBKB (209342_s_at 209341_s_at 211027_s_at), IKBKG
(209929_s_at 36004_at), ILlORA (204912_at), IL12RB1 (206890_at
239522_at 1552584_at), IL12RB2 (206999_at), IL13RA1 (210904_s_at
201888_s_at 211612_s_at 201887_at), IL15RA (207375_s_at), IL17RD
(227997_at 229263_at), IL21R (237753_at 221658_s_at 219971 _at),
IL23R (1561853_a_at 1552912_a_at), IL2RB (205291_at), IL2RG
(204116_at), IL3RA (206148_at), IL4R (203233_at), IL5RA (211516_at
211517_s_at 207902_at 210744_s_at), IL6R (205945_at 217489_s_at),
IL6ST (211000_s_at 212196_at 234474_x_at 214077_x_at 204863_s_at
212195_at 234967_at 204864_s_at), IL7R (226218_at 205798_at), IL9R
(217212_s_at 208164_s_at), IMPDH2 (201892_s_at), INPP5D (1568943_at
233545_at 203331_s_at 203332_s_at), INPPL1 (201598_s_at), INS
(206598_at), INSR (227432_s_at 226450_at 213792_s_at 243002_at
226212_s_at 226216_at 207851_s_at), INSRR (215776_at), IQGAP1
(213446_s_at 210840_s_at 200791_s_at), IRAK1 (201587_s_at
1555784_s_at), IRS1 (238933_at 242979_at 239463_at 235392_at
204686_at), IRS2 (236338_at 209185_s_at 209184_s_at), IRS4
(207403_at), ITFG2 (226295_at 220589_s_at), ITGA5 (201389_at),
ITGAV (202351_at), ITGB1 (1553530_a_at 211945_s_at 1553678_a_at
215879_at 216190_x_at 216178_x_at), ITGB2 (1555349_a_at
202803_s_at), ITGB3 (204625_s_at 204627_s_at 216261_at 211579_at
215240_at 204628_s_at 204626_s_at), ITGB4 (204989_s_at 214292_at
230704_s_at 211905_s_at 204990_s_at), ITGB5 (201125_s_at
214021_x_at), ITGB6 (208084_at 208083_s_at), ITK (211339_s_at),
ITPR1 (1562373_at 203710_at 231329_at 216944_s_at 244090_at
211323_s_at), ITSN1 (2092979_at 209298_s_at 210713_at 35776_at
207322_at), JAK1 (1552611_a_at 239695_at 1552610_a_at 201648_at),
JAK2 (205842_s_at 205841_at 1562031_at), JAK3 (211109_at
211108_s_at 207187_at 227677_at), JAKMIP1 (238600_at), JUN
(201466_s_at 201464_x_at 213281_at 201465_s_at), JUP (201015_s_at),
KALRN (232717_at 206078_at 227750_at 236651_at 205635_at), KCNA2
(239118_at 208564_at), KCNAS (206762_at), KCNB1 (211006_s_at),
KCNMA1 (221584_s_at 228414_at 221583_s_at 214921_at), KCNQS
(244623_at 223891_at), KDR (203934_at), KHDRBS1 (200040_at
201488_x_at), KIAA1377 (232166_at 236325_at 235956_at), KIDINS220
(1557246_at 214932_at 212163_at 212162_at), KIFAP3 (203333_at),
KIR2DL3 (211410_x_at 211397_x_at 216907_x_at 211532_x_at
207314_x_at 208122_x_at 208203_x_at 216552_x_at 211688_x_at
208179_x_at 211687_x_at 207313_x_at 216676_x_at 208198_x_at
210890_x_at), KIT (205051_s_at), KITLG (211124_s_at 226534_at
207029_at), KL (205978_at), KPNA2 (201088_at 211762_s_at), KPNB1
(217027_x_at 208975_s_at 213507_s_at 208974_x_at), KRAS
(1559204_x_at 1559203_s_at 204009_s_at 214352_s_at), KRT17
(212236_x_at 205157_s_at), KRT18 (201596_x_at), KRT27 (240388_at),
KRT7 (1558394_s_at 209016_s_at), KRT8 (216568_x_at 230116_at
209008_x_at), KRTAP4-12 (224269_at), LAIR1 (210644_s_at
208071_s_at), LAT (209881_s_at 211005_at), LAX1 (207734_at), LCK
(204891_s_at 204890_s_at), LCP2 (205270_s_at 205269_at), LCT
(206945_at), LDLRAP1 (221790_s_at 57082_at), LEPR (209894_at
1556919_at 211356_x_at 211354_s_at 207255_at 211355_x_at), LIME1
(219541_at), LMO4 (229537_at 241922_at 209204_at 227155_at
209205_s_at), LMTK2 (235307_at 206223_at 226375_at), LMTK3
(1557103_a_at), LPHN1 (219145_at 47560_at 203488_at), LPXN
(216250_s_at 242778_at), LRP1 (200785_s_at 1555353_at 200784_s_at),
LRPPRC (211615_s_at 211971_s_at 230194_at 1557360_at 230594_at),
LRSAM1 (227675_at 235449_at), LTK (207106_s_at 217184_s_at), LYN
(202625_at 210754_s_at 202626_s_at), MAD2L1 (1554768_a_at
203362_s_at), MAG (217447_at 216617_s_at), MAGED1 (209014_at
244878_at), MAP2 (241044_x_at 210015_s_at 225540_at), MAP2K1
(202670_at), MAP3K1 (225927_at 214786_at), MAP3K14 (205192_at),
MAP3K3 (227131_at 203514_at), MAP3K4 (204089_x_at 216199_s_at),
MAP3K5 (203837_at 203836_s_at), MAP4K1 (214339_s_at 214219_x_at
206296_x_at), MAP4K5 (211081_s_at 203553_s_at 203552_at), MAPK1
(208351_s_at 224621_at 1552264_a_at 1552263_at 229847_at
212271_at), MAPK14 (202530_at 210449_x_at 211561_x_at 211087_x_at),
MAPK15 (241357_at), MAPK3 (212046_x_at), MAPK8 (226046_at 226048_at
210671_x_at 210477_x_at), MAPK8IP1 (213014_at 213013_at), MAPK8IP2
(208603_s_at 205050_s_at), MAPK8IP3 (216139_s_at 213177_at
230162_s_at 213178_s_at 232085_at 216137_s_at), MAPK9 (210570_x_at
225781_at 203218_at), MAPT (203929_s_at 203930_s_at 225379_at
206401_s_at 203928_x_at), MATK (206267_s_at), MAX (209331_s_at
209332_s_at 210734_x_at 208403_x_at 214108_at), MCAM (210869_s_at
209086_x_at 211340_s_at 209087_x_at), MCF2 (1555313_a_at
217004_s_at 208017_s_at), MDK (209035_at), MDM2 (217542_at
229711_s_at 225160_x_at 217373_x_at 244616_x_at 205386_s_at
211832_s_at), MERTK (211913_s_at 206028
.sub.-s_at), MET (213807_x_at 203510_at 211599_x_at 213816_s_at),
MICAL1 (218376_s_at), CLNK (1570239_a_at 1562587_at), MLLT4
(238871_at 230622_at 208512_s_at), MME (203434_s_at 203435_s_at),
MMP16 (207012_at 223614_at 208167_s_at 208166_at 207013_s_at), MMP2
(201069_at), MPDZ (205079_s_at 213306_at), MPL (211903_s_at
216825_s_at 207550_at), MPZL1 (210594_x_at 201874_at 210210_at
201875_s_at 210087_s_at 231621_at), MS4A1 (228592_at 228599_at
210356_x_at 217418_x_at), MS4A2 (207497_s_at 207496_at), MSN
(200600_at 240960_at), MST1 (216320_x_at 205614_x_at 213380_x_at),
MST1R (205455_at), MUC1 (211695_x_at 207847_s_at 213693_s_at),
MUC20 (231941_s_at 230043_at 226622_at), MUC4 (204895_x_at
235055_x_at 217109_at 217110_s_at), MUSK (207632_at 207633_s_at
241122_s_at), MYC (202431_s_at), MYD88 (209124_at), MYLK
(1568770_at 1563466_at 2248239_at 202555_s_at), NCAM1 (214952_at
209968_s_at 229799_s_at 227394_at 217359_s_at 212843_at), NCF1
(204961_s_at 214084_x_at), NCK1 (211063_s_at 204725_s_at
229895_s_at), NCK2 (203315_at), NCOA3 (207700_s_at 209061_at
211352_s_at 209062_x_at 209060_x_at), NCOA6 (208979_at 1568874_at),
NCOR1 (234313_at 200854_at 200856_x_at 200855_at 200857_s_at),
NCSTN (237076_at 208759_at), ND2 (not found), NDUFS1 (203039_s_at
229647_at 236356_at), NDUFS6 (203606_at), NEDD4 (213012_at), NEDD4L
(226974_at 212445_s_at 241396_at 212448_at 236490_at), NEDD8
(243733_at 201840_at), NEDD9 (1569020_at 202150_s_at 202149_at),
NEO1 (204321_at 229877_at 225270_at), NF2 (204991_s_at 238618_at
217150_s_at 211091_s_at 210767_at 218915_at 211092_s_at
211017_s_at), NFAM1 (243099_at 230322_at), NFKBIA (201502_s_at),
NFKBIB (214448_x_at 228388_at 214062_x_at), NGEF (227240_at
243556_at), NGF (206814_at), NGFR (205858_at), NGFRAP1
(217963_s_at), NMT1 (201159_s_at 201157_s_at 201158_at), NOS2
(210037_s_at), NOTCH1 (218902_at), NPHP1 (206285_at 238844_s_at
238843_at), NPHS1 (207673_at 241181_x_at), NPM1 (200063_s_at
221923_s_at 221691_x_at), NR3C1 (201866_s_at 232431_at 216321_s_at
201865_x_at 211671_s_at), NRAS (224985_at 202647_s_at), NRG1
(208231_at 208232_x_at 208241_at 208230_s_at 206237_s_at
206343_s_at), NRG2 (242303_at 208062_s_at 206879_s_at), NRG3
(229233_at), NRG4 (242426_at), NRP1 (210510_s_at 212298_at), NRP2
(223510_at 211844_s_at 210842_at 229225_at 1555468_at 230410_at
210841_s_at 214632_at 228102_at 225566_at 228103_s_at), NRTN
(210683_at), NTF3 (206706_at), NTF4 (not found), NTRK1
(208605_s_at), NTRK2 (214680_at 207152_at 236095_at 221796_at
229463_at 221795_at), NTRK3 (217033_x_at 217377 _x_at 215115_x_at
215025_at 206462_s_at 1557795_s_at 228849_at), NUMB (209073_s_at
230462_at 207545_s_at), NUMBL (242195_x_at 224059_s_at), NXF1
(208922_s_at), OCLN (235937_at 209925_at), ODF2L (230926_s_at
237420_at 228577_x_at), ONECUT1 (2107459_at), OSMR (1554008_at
226621_at 205729_at), P2RY2 (206277_at), PA2G4 (214794_at
208676_s_at), PAG1 (225626_at 227354_at 225622_at), PAK1 (226507_at
209615_s_at), PAK2 (208878_s_at 208877_at 208876_s_at 236283_x_at
208875_s_at 205962_at 1559052_s_at 244268_x_at), PAK4 (203154_s_at
33814_at 215326_at), PARD3 (221526_x_at 221527_s_at 221280_s_at
210094_s_at), PAX3 (207679_at 231490_at 231666_at 216059_at
207680_x_at), NAMPT (217739_s_at 217738_at 1555167_s_at), PCBD2
(1554894_a_at 223712_at), PDAP1 (202290_at 217624_at), PDE4A
(211901_s_at 211591_s_at 204735_at 211447_s_at), PDE4D (228962_at
1554717_a_at 243586_at 204491_at 222322_at 210837_s_at 210836_x_at
211840_s_at), PDE6G (210060_at), PDGFA (229830_at 216867_s_at
205463_s_at), PDGFB (204200_s_at 216061_x_at 217112_at), PDGFC
(242171_at 222719_s_at 218718_at), PDGFD (222860_s_at 219304_s_at),
PDGFRA (211533_at 237696_at 1554828_at 203131_at 215305_at), PDGFRB
(202273_at), PDLIM7 (203369_x_at 214266_s_at 214121_x_at
203370_s_at 214122_at), PDPK1 (221244_s_at 232050_at 204524_at
32029_at 224986_s_at 222260_at), PECAM1 (1558397_at 208983_s_at
208982_at 208981_at), PELP1 (215354_s_at), PGF (209652_s_at), PGR
(208305_at 228554_at), PIAS1 (217864_s_at 222371_at), PICK1
(204746_s_at), PIK3AP1 (1554508_at 226459_at), PIK3C2A
(1569022_a_at 235792_x_at 1553694_a_at 213070_at 1569021_at
226094_at), PIK3C2B (204484_at), PIK3CA (204369_at), PIK3CB
(212688_at 217620_s_at), PIK3CG (206369_s_at 206370_at), PIK3R1
(212240_s_at 212249_at 212239_at), PIK3R2 (229392_s_at 207105_s_at
1568629_s_at), PIK3R3 (202743_at 211580_s_at), PILRB (225321_s_at
220954_s_at), PIM1 (209193_at), PIP4K2A (205570_at 229713_at
212829_at), PIP4K2B (1553047_at 201081_s_at 1553048_a_at
201080_at), PIP4K2C (218942_at), PIP5K1A (210256_s_at 207391_s_at
211205_x_at), PIP5K1B (205632_s_at), PIP5K1C (212518_at), PITPNA
(241974_at 201191_at 239124_at 201192_s_at 201190_s_at 237424_at),
PITPNM1 (203826_s_at), PITPNM2 (1552923_a_at 1552924_a_at
232950_s_at), PITPNM3 (221254_s_at 230076_at), PKD1 (216949_s_at
202327_s_at 202328_s_at), PKIA (204612_at), PLA2G4A (210145_at),
PLAUR (210845_s_at 214866_at 211924_s_at), PLCE1 (205112_at
205111_s_at), PLCG1 (216551_x_at 202789_at), PLCG2 (204613_at),
PLD1 (215723_s_at 226636_at 177_at 205203_at 1557126_a_at
215724_at), PLD2 (209643_s_at), PLEC (216971_s_at 201373_at
238083_at), PLK4 (204887_s_at 204886_at 211088_s_at), PLSCR1
(202446_s_at 202430_s_at), PLXNA1 (1558140_at 221537_at
221538_s_at), PLXNB1 (215807_s_at 215668_s_at), PMAIP1 (204286_s_at
204285_s_at), POLA2 (204441_s_at), POLR2A (202725_at 217420_s_at
217415_at), POLR2I (212955_s_at), POU2AF1 (205267_at), PPARD
(210636_at 37152_at 208044_s_at), PPIA (217602_at 211378_x_at
211978_x_at 211765_x_at 201293_x_at 226336_at 212661_x_at), PPP1CB
(201409_s_at 201408_at 201407_s_at 228222_at), PPP1R15A (37028_at
202014_at), PPP1R2 (202165_at 202166_s_at 213774_s_at), PPP1R8
(207830_s_at), PPP2CA (208652_at), PPP2R1B (202886_s_at 202885_s_at
202883_s_at 202884_s_at 222351_at), PPP2R5A (202187_s_at), PRAM1
(241742_at), PRDX2 (215067_x_at 39729_at), PRKACA (202801_at
216234_s_at), PRKAR1A (242482_at 200604_s_at 200605_s_at
200603_at), PRKAR2A (204842_x_at 204843_s_at), PRKCA (213093_at
1560074_at 215195_at 206923_at 215194_at), PRKCB (227817_at
209685_s_at 207957_s_at 230437_s_at 228795_at 227824_at), PRKCD
(202545_at), PRKCE (206248_at 226101_at), PRKCH (218764_at
206099_at), PRKCI (213518_at 209678_s_at 209677_at), PRKCQ
(210039_s_at 210038_at), PRKCZ (202178_at), PRKD1 (205880_at
217705_at), PRKDC (208694_at 210543_s_at), PRLR (211917s_at
231981_at 243755_at 216638_s_at 227629_at 210476_s_at 206346_at),
PRMTS (1564520_s_at 1564521_x_at 217786_at), PROS1 (207808_s_at),
PSMA4 (203396_at), PSMD13 (201233_at 201232_s_at), PSTPIP1
(211178_s_at), PTAFR (206278_at 211661_x_at 227184_at), PTEN
(233314_at 233254_x_at 211711_s_at 225363_at 227469_at 217492_s_at
204054_at 204053_x_at), PTGES3 (200627_at), PTK2 (1559529_at
208820_at 207821_s_at 241453_at), PTK2B (203110_at 203111_s_at),
PTK6 (1553114_a_at), PTK7 (1555324_at 207011_s_at), PTN
(211737_x_at 209466_x_at 209465_x_at), PTPN1 (202716_at 240260_at
239526_x_at 217689_at), PTPN11 (209895_at 209896_s_at 205867_at
212610_at 205868_s_at 241930_x_at 1552637_at), PTPN12 (216915_s_at
202006_at 216884_at 244356_at), PTPN18 (203555_at 213521_at
1569552_at), PTPN2 (213136_at 204935_at 213137_s_at), PTPN21
(205438_at 226380_at 222092_at 40524_at 1320_at), PTPN22 (236539_at
208010_s_at 206060_s_at), PTPN3 (227944_at 203997_at), PTPNS
(236456_at 233471_at), PTPN6 (206687_s_at), PTPRA (213799_s_at
213795_s_at), PTPRB (205846_at 217177_s_at 230250_at), PTPRC
(212587_s_at 207238_s_at 1552480_s_at 1569830_at 212588_at), PTPRE
(221840_at 1559018_at), PTPRF (215066_at 200637_s_at 200636_s_at
200635_s_at), PTPRH (208300_at), PTPRJ (210173_at 214137_at
227396_at), PTPRO (208121_s_at 1554199_at), PTPRS (1555666_at
210823_s_at 229465_s_at 226571_s_at), PTPRU (211320_s_at), PTPRZ1
(204469_at), PXN (211823_s_at 201087_at), RAD51 (205023_at
205024_s_at), RAD52 (205647_at 210630_s_at 211904_x_at), RAD9A
(204828_at 1562022_s_at), RADIL (223693_s_at), RAF1 (201244_s_at),
RALA (214435_x_at 224880_at), RALB (202101_s_at 202100_at), RALGDS
(209050_s_at 209051_s_at), RAN (200750_s_at), RANBP10 (53987_at
221809_at 1558773_s_at), RANBP9 (202582_s_at 216125_s_at
202583_s_at), RAP1A (1555339_at 202362_at 1555340_x_at), RAPGEF1
(225738_at 226389_s_at 204543_at), RAPSN (211570_s_at), RASA1
(210621_s_at 202677_at), RASA3 (225562_at 206221_at 206220_s_at),
RASA4 (212706_at 208534_s_at 212707_s_at), RASGEF1C (236748_at),
RASGRF1 (214905_at 215688_at 1554992_at 210550_s_at), RB1
(203132_at 211540_s_at), RB1CC1 (202033_s_at 237626_at
202034_x_at), RELA (230202_at 201783_s_at 209878_s_at), RET
(215771_x_at 211421_s_at 205879_x_at), RFX1 (206321_at 226786_at),
RGS16 (209324_s_at 209325_s_at), RGS2 (202388_at), RGS4 (204337_at
204339_s_at 204338_s_at), ARHGAP32 (242196_at 203431_s_at
210791_s_at 229648_at), RIN1 (205211_s_at), RIN2 (209684_at
233811_at), RIPK1 (209941_at 226551_at), RIT1 (239843_at
243463_s_at 209882_at), RIT2 (206984_s_at), RNF130 (217865_at),
RNF41 (201961_s_at 201962_s_at), ROBO1 (213194_at 240558_at), ROCK1
(235854_x_at 214578_s_at 230239_at 213044_at), ROR1 (205805_s_at
211057_at), ROR2 (231000_at 205578_at), ROS1 (207569_at), RPL10
(200724_at 221989_at 200725_x_at), RPL18A (200869_at), RPL8
(200936_at), RPLP2 (200908_s_at 200909_s_at), RPN1 (201011_at),
RPS6KA1 (203379_at), RPS6KA2 (240720_at 236658_at 212912_at
204906_at 1557970_s_at), RRAS (212647_at), RTN1 (210222_s_at
203485_at), RTN3 (219549_s_at 224564_s_at), RUFY1 (218243_at
233380_s_at), RUFY2 (1554133_at 238550_at 1569630_a_at 233192_s_at
235345_at), RUNX1 (236114_at 209359_x_at 211182_x_at 211181_x_at
209360_s_at 210805_x_at 211180_x_at 208129_x_at 211620_x_at
210365_at), RUSC1 (206949_s_at), RYBP (201846_s_at 242719_at
201844_s_at 237456_at 201845_s_at), RYK (214172_x_at 202853_s_at
216976_s_at 238210_at), S100A7 (205916_at), S100A9 (203535_at),
SAT1 (210592_s_at 213988_s_at 203455_s_at), SCAMPI (212425_at
1552978_a_at 206668_s_at 212416_at 206667_s_at 212417_at), SCAMP3
(201771_at), SDC2 (212154_at 212158_at 212157_at), SDC3
(1554864_a_at 202898_at), SDCBP (200958_s_at), SEC13 (207707_s_at),
SELE (206211_at), SELPLG (209879_at 209880_s_at), SERPINA3
(202376_at), SEZ6 (243430_at 229651_at), SF3B3 (200688_at
200687_s_at), SF3B4 (209044_x_at), SFN (33323_r at 33322_i at
209260_at), SRSFS (210077_s_at 203380_x_at 212266_s_at), SGSM2
(212319_at 36129_at), SH2B1 (209322_s_at 40149_at), SH2B2
(205367_at), SH2B3 (203320_at), SH2D1A (211210_x_at 211209_x_at
211211_x_at 210116_at), SH2D1B (1553177_at 1553176_at), SH2D2A
(207351_s_at), SH2D3A (219513_s_at 222169_x_at), SH2D3C (226673_at
1552667_a_at), SH3BGRL (201312_s_at 201311_s_at), SH3BGRL3
(221269_s_at), SH3BP1 (215799_at 213633_at), SH3BP2 (211250_s_at
209370_s_at 209371_s_at), SH3BP5 (201811_x_at 201810_s_at), SH3GL2
(205751_at), SH3KBP1 (1554168_a_at 235692_at 223082_at), SH3PXD2A
(224817_at 213252_at 207661_s_at), SHB (204657_s_at 230459_s_at
204656_at 1557458_s_at 243595_at), SHC1 (201469_s_at 214853_s_at),
SHC2 (213464_at), SHC3 (243881_at 206330_s_at 229824_at), SHD
(227845_s_at), SHE (229910_at), SHF (228922_at), SIRPA (202897_at
202896_s_at 202895_s_at 217024_x_at), SIT1 (205484_at), SIVA1
(210792_x_at 203489_at), SKAP1 (205790_at), SKAP2 (216899_s_at
204362_at 204361_s_at 225639_at), SLA (203761_at 203760_s_at), SLA2
(232234_at 1555688_s_at), SLAMF1 (1555626_a_at 206181_at), SLAMF6
(1552497_a_at), SLC25A6 (212085_at 212826_s_at), SLC2A1 (235633_at
201250_s_at 201249_at), SLC3A2 (200924_s_at), SLC4A1 (205592_at
1552713_a_at), SLC9A2 (211116_at), SLC9A3R1 (201349_at), SLC9A3R2
(209830_s_at), SMN1 (242495_at 203852_s_at), SMAD2 (239271_at
226563_at 243895_x_at 203076_s_at 203075_at 235598_at 203077_s_at),
SMAD4 (1565702_at 202527_s_at 235725_at 202526_at 235622_at),
SMARCB1 (212167_s_at), SMARCE1 (229511_at 211989_at 211988_at),
SMC1A (1555677_s_at 239688_at 217555_at 201589_at), SMG7 (242575_at
217189_s_at 201793_x_at 201794_s_at), SMURF2 (232020_at 230820_at
227489_at 205596_s_at), SNAP29 (239084_at 222597_at 218327_s_at),
SNAPIN (223066_at), SNCA (211546_x_at 207827_x_at 236081_at
204467_s_at 204466_s_at), SNRPD2 (200826_at), SNTB2 (227312_at
226685_at 213814_s_at 205314_x_at 238925_at 205315_s_at), SNURF
(206042_x_at 201522_x_at), SNX1 (213364_s_at 201716_at 214531_s_at
216357_at), SNX2 (202113_s_at 202114_at), ARHGAP33 (233885_at
215256_x_at 213827_at), SNX4 (205329_s_at 212652_s_at), SNX6
(217789_at 222410_s_at), SNX9 (223028_s_at 223027_at), SO C S1
(210001_s_at 209999_x_at 210000_s_at 213337_s_at), SOCS2 (203373_at
203372_s_at), SOCS3 (214105_at 206360_s_at 206359_at 227697_at),
SOCSS (208127_s_at 209648_x_at 209647_s_at), SOCS6 (227542_at
206020_at 214462_at), SORBS1 (237026_at 211705_s_at 211819_s_at
222513_s_at 218087_s_at), SORBS2 (1558815_at 237285_at 241104_at
225728_at 240120_at 204288_s_at 220858_at), SO S1 (1557354_at
212777_at 242018_at 242682_at 229261_at 227426_at 212780_at), SOS2
(217575_s_at 217644_s_at 217576_x_at 212870_at 211665_s_at), SPHK1
(219257_s_at), SPHK2 (209857_s_at 40273_at), SPN (216980_s_at
216981_x_at 206057_x_at 206056_x_at 1568964_x_at), SPRED1
(244439_at 235074_at 226837_at), SPRED2 (212466_at 212458_at
214026_s_at), SPSB1 (226075_at 219677_at), SPTAN1 (214926_at
215235_at 214925_s_at 208611_s_at), SQSTM1 (217252_at 244804_at
201471_s_at 217255_at 213112_s_at), SRC (1565082_x_at 237103_at
213324_at 1565080_at 221284_s_at 1558211_s_at), SRF (202401_s_at
202400_s_at), SRM (201516_at), ST5 (202440_s_at), STAM
(203544_s_at), STAM2 (228254_at 215044_s_at 208194_s_at 242569_at
209649_at), STAP1 (220059_at 1554343_a_at), STAP2 (221610_s_at),
STAT1 (AFFX-HUMISGF3A/M97935_5_at AFFX-HUMISGF3A/M97935_3_at
200887_s_at AFFX-HUMISGF3A/M97935_MB at AFFX-HUMISGF3A/M97935_MA at
209969_s_at), STAT2 (225636_at 205170_at 217199_s_at), STAT3
(208991_at 225289_at 208992_s_at 243213_at), STATSA (203010_at),
STATSB (1555086_at 212550_at 205026_at 1555088_x_at 212549_at),
STATE (201332_s_at 201331_s_at), STK39 (202786_at), STUB1
(217934_x_at 233049_x_at 227625_s_at), SUPT4H1 (201484_at
201483_s_at), SUPT6H (208830_s_at 208420_x_at 1554311_a_at
208831_x_at), SYK (226068_at 209269_s_at 207540_s_at 244023_at),
SYN1 (221914_at 1553264_a_at), SYNCRIP (217834_s_at 209025_s_at
209024_s_at 217833_at 217832_at 1555427_s_at), SYNE1 (244070_at
215350_at 209447_at 244144_at), SYNGAP1 (230297_x_at 234285_at),
SYNJ1 (212990_at 232993_at 207594_s_at), TAF1 (227205_at 216955_at
216711_s_at), TEC (206301_at), TEK (217711_at 206702_at), TENC1
(212494_at), TERT (207199_at 1555271_a_at), TGFA (211258_s_at
205016_at 205015_s_at), TGFB1I1 (209651_at), TGFBR1 (224793_s_at
206943_at), TH1L (225006_x_at 225865_x_at 225261_x_at 220607_x_at),
THOCS (209418_s_at 209419_at), THY1 (213869_x_at 208850_s_at
208851_s_at), TIAF1 (202039_at), TIAM1 (213135_at 206409_at), TIE1
(1560657_at 204468_s_at), TIMP3 (201149_s_at 201150_s_at
201147_s_at 201148_s_at), TIRAP (1554091_a_at 239796_x_at 236687_at
1552360_a_at 1552804_a_at), TJP1 (214168_s_at 202011_at), TLN1
(203254_s_at), TLR2 (204924_at), TLR3 (206271_at 239587_at), TLR4
(221060_s_at 1552798_a_at 224341_x_at 232068_s_at), TLR6 (239021_at
207446_at), TLR8 (220832_at 229560_at), TLR9 (223903_at), TM4SF1
(209387_s_at 238168_at 209386_at 215033_at 215034_s_at), TMF1
(215855_s_at 227685_at 214948_s_at 235566_at 213024_at), TNC
(201645_at), TNFAIP1 (201208_s_at 201207_at), TNFRSF10A
(1552648_a_at), TNFRSF11A (207037_at 238846_at), TNFRSF1A
(207643_s_at), TNFRSF1B (203508_at), TNFRSF8 (206729_at), TNFSF11
(211153_s_at 241248_at 210643_at), TNIP2 (48531_at 218335_x_at
232160_s_at), TNK1 (217149_x_at 205793_x_at), TNK2 (203839_s_at
216439_at 1555557_a_at 228279_s_at 203838_s_at), TNS1 (221748_s_at
218863_s_at 221246_x_at 221747_at 218864_at), TNS4 (230398_at
222265_at), TOB1 (228834_at 202704_at), TOM1L1 (240261_at
204485_s_at), TP53 (211300_s_at 201746_at), TP53BP2 (203120_at),
TP53RK (225402_at 235192_at), TP73 (1554379_a_at 232546_at
220804_s_at), TRAF1 (235116_at 205599_at), TRAF2 (204413_at), TRAF3
(208315_x_at 221571_at), TRAF6 (205558_at), TRAT1 (217147_s_at),
TRIO (240399_at 209010_s_at 209011_at 209012_at 244527_at
209013_x_at 240773_at 208178_x_at), TRIP6 (209129_at), TRPC6
(217287_s_at 241558_at 206528_at), TRPV4 (219516_at), TSG101
(230176_at 201758_at), TSHR (215443_at 215442_s_at 210055_at), TTR
(209660_at), TUB (208431_s_at 228882_at 210737_at), TUBA1A
(211072_x_at 211058_x_at 201090_x_at 209118_s_at 211750_x_at
212639_x_at 209251_x_at 213646_x_at), TUBA1B (211072_x_at
211058_x_at 201090_x_at 211750_x_at 212639_x_at 209251_x_at
213646_x_at), TUBA3C (210527_x_at 216323_x_at), TUBA4A (212242_at),
TUBB (212320_at 211714_x_at 209026_x_at), TUBB2A (213476_x_at
204141_at 209372_x_at), TXK (206828_at), TYK2 (205546_s_at), TYRO3
(211432_s_at 211431_s_at 1566934_at), TYROBP (204122_at), UBASH3B
(244068_at 240387_at 238587_at 238462_at 228353_x_at 228359_at),
UBB (200633_at 242300_at), UBE2L3 (200684_s_at 200683_s_at
200682_s_at 200676_s_at), UBE3A (211575_s_at 213128_s_at
211285_s_at 213291_s_at 212278_x_at), UCP2 (208998_at 208997_s_at),
UNC119 (203271_s_at), UQCRB (209066_x_at 209065_at 205849_s_at
244293_at), VAV1 (206219_s_at), VAV2 (205537_s_at 226063_at
205536_at), VAV3 (218806_s_at 224221_s_at 218807_at), VCL
(200930_s_at 200931_s_at), VCP (208649_s_at 228442_at 208648_at),
VDR (204255_s_at 204254_s_at 204253_s_at 213692_s_at), VEGFA
(212171_x_at 211527_x_at 210512_s_at 210513_s_at), VEGFB
(203683_s_at), VEGFC (209946_at), VIL1 (1554943_at 1554945_x_at
205506_at), VTI1B (209452_s_at 225926_at), WAS (205400_at 38964_r
at), WASF1 (204165_at), WASF2 (224563_at 224562_at 221725_at), WASL
(224813_at 205810_s_at 230340_s_at 205809_s_at), WBP11 (217822_at
217821_s_at), SNRNP40 (215905_s_at), WIPF1 (202664_at 231182_at
202665_s_at 202663_at), WISP2 (205792_at), WNT1 (208570_at), WNT3A
(not found), WNTSA (213425_at 205990_s_at 231227_at), WT1
(216953_s_at 206067_s_at), WWOX (237035_at 223868_s_at 219077_s_at
221147_x_at 210695_s_at 242099_at 223747_x_at), XPO1 (208775_at),
XPO6 (211982_x_at 214784_x_at), XRCCS (208643_s_at 208642_s_at),
XRCC6 (200792_at), YAP1 (224895_at 224894_at 213342_at), YES1
(202932_at 202933_s_at).
EXAMPLE 5
Proximity Ligation Assay for Egfr and Grb2 Interaction In Cell
Lines Using Intracellular Egfr EpitopE
Updated Methods
Reagent Information:
Antibodies:
[0244] mGRb2 (Mouse monoclonal, clone 81 (Cat# 610112, BD
Biosciences)
[0245] rEGFR (Rabbit monoclonal, clone D38B1 (Cat# 4267, Cell
Signaling)
[0246] Working dilutions: m-EGFR (1:50) +r-GRb2 (1:50)
Solutions:
[0247] .sub.MQH.sub.2O
[0248] PBS
[0249] Kit Components: [0250] Secondary antibody-PLA probes (plus
and minus) [0251] Ligation reagents [0252] Amplification ligation
reagents [0253] 1.times. Wash Buffer B [0254] 0.01.times.Wash
Buffer B [0255] Mounting media (w/DAPI)
Experimental Procedure:
[0256] 1. Plate cells in chamber wells 24 h before processing. For
most cell lines, 50.times.10.sup.4 per well (0.8cm.sup.2) is
sufficient. This number should be adjusted for longer culture times
and different-sized chamber wells.
[0257] 2. Wash each well with PBS (2.times.2 m).
[0258] 3. Fix cells by adding 400 .mu.l 10% Buffered formalin per
well. Incubate 20 m RT with gentle rocking.
[0259] 4. Wash each well with PBS (3.times.2 m).
[0260] 5. Permabilize cells by adding 0.5% Triton X100 (diluted in
PBS). Incubate 10 m RT, no rocking.
[0261] 6. Wash each well with PBS (3.times.2m).
[0262] 7. Remove chamber from slide. Note that excess silicone
adhesive should be removed with a scalpel to ensure that it does
not interfere with coverslipping later.
[0263] 8. Add 1.5% BSA (diluted in PBS) using a volume sufficient
to fully cover each well. For 8-well slides (0.8cm.sup.2), a volume
of 50 .mu.l is sufficient. A grease pen can be used to ensure a
hydrophopic barrier exists between wells, but is usually not
necessary. Incubate 30m RT in humidity chamber.
[0264] 9. Primary antibodies: Add 50 .mu.l per well appropriate
primary antibody soultuion [r-EGFR and m-GRb2] diluted in PBS.
Incubate ON in humidity chamber 4.degree. C. with gentle
rocking.
[0265] a. Record start time
[0266] b. Incubation time should be 16-18 hours
[0267] 10. Next day: Tap off primary antibody solution. Wash 2
times with sequential immersion in PBS in Coplin jars. Wash
2.times.5 m RT with gentle rocking.
[0268] 11. PLA probes (secondary antibody-DNA conjugates): Mix and
dilute the two PLA probes 1:5 (diluted in PBS) (use 8 .mu.l minus
probe and 8 .mu.l probe plus and 24 .mu.l PBS=40 .mu.l/cm.sup.2).
Ensure complete coverage of tissue area. Incubate slides 1 h
37.degree. C. in pre-heated humidity chamber.
[0269] 12. Tap off PLA probes and wash. Wash 2.times.5 m with PBS,
RT with gentle rocking. Thaw 5.times. ligation mix at beginning of
washes.
[0270] 13. PLA probe ligation: Dilute ligation stock 1:5 in
.sub.MQH.sub.2O while waiting, saving room for ligase (e.g., 8
.mu.l 5.times. ligation mix and 31 .mu.l .sub.MQH.sub.2O=39 .mu.l).
Add Ligase into ligation solution 1:40 during final wash above.
Cover tissue area completely using 40 .mu.l/cm.sup.2. Incubate for
30 m 37.degree. C. in pre-warmed humidity chamber.
[0271] 14. Tap off ligation solution and wash. Wash 2.times.2 m RT
with gentle rocking (PBS). Thaw 5.times. amplification mix at
beginning of washes (avoiding light).
[0272] 15. PLA probe amplification-hybridization-fluorescence:
Dilute Amplification stock 1:5 in MQH2O while waiting (8 .mu.l and
31.5 .mu.l .sub.MQH.sub.2O=39 .mu.l). Add Polymerase 1:80 into
amplification solution then add polymerase-amplification solution.
Cover tissue area completely using 40 .mu.l/cm.sup.2. Incubate 2 h
37.degree. C. pre-warmed humidity chamber (avoid light).
[0273] 16. Tap off amplification solution and wash 2.times.10 m RT
with 1.times. Wash Buffer B. Rinse slide in 0.01.times. Wash Buffer
B.
[0274] 17. Allow slides to dry for .about.10 m in dark. Mount with
Invitrogen Prolong Gold with DAPI. Add mounting media to cover slip
and gently press down onto tissue area. Allow to dry overnight and
image by confocal microscopy next day. Note that Prolong Gold is an
aqueous-based mounting media and will not cure. Care should be
taken to avoid moving coverslip, once mounted. Store slides at
4.degree. after initial overnight dry.
[0275] 18. Confocal Microscopy: Use the Leica TCS SP5 laser
scanning confocal microscope. Place slide on scope and find best
in-focus plane. Take care to avoid imaging along edges of tissue,
whenever possible. Note that Cy5 foci cannot be seen through the
confocal eyepiece due to the far red wavelength. Use 40.times. oil
objective and begin with voltage settings of .about.450 for the 405
laser line (DAPI) and .about.580 for the 633 laser line (Cy5). Use
1024.times.1024 image size and acquire at 200 Hz.
[0276] 19. Use the "live" scan to observe foci. Use the z-plane
wheel to scroll above and below the focus plane until foci
disappear and only DAPI remains, set upper and lower limits.
Generally, 12 z-slices at 0.76 .mu.m provides sufficient coverage
of all foci.
[0277] 20. Do not adjust 633 laser line between samples. DAPI can
be adjusted and will vary among cell types. Keep DAPI relatively
dim as the merged images may be overexposed if DAPI is too
intense.
[0278] 21. After collecting samples (2-3 fields of view for each
sample), process each series as a maximum projection. These images
can then be exported as TIFF files.
EXAMPLE 6
Duolink Assay for Egfr and Grb2 Interaction from Ffpe Tissues With
Cytokeratin Counterstaining
Reagent Information
Antibodies:
[0279] GRB2 (Mouse monoclonal, clone 81 (Cat# 610112, BD
Biosciences)
[0280] EGFR (Rabbit monoclonal, clone D38B1 (Cat#4267, Cell
Signaling)
[0281] Cytokeratin, pan (Guinea Pig polyclonal (Cat# 9 9097-48B
097-48B, US Biological)
[0282] AlexFluor555, goat anti-guinea pig (Invitrogen,
Cat#A-21435)
[0283] Working dilutions: m-EGFR (1:100)+r-GRb2 (1:100)+gp-CK
(1:100)
Solutions:
[0284] AR Buffer (PT4) (10 mM Tris Base, 1 mM EDTA, .05% Tween 20,
pH9) PBS
[0285] .sub.MQH.sub.2O
[0286] 0.05% PB ST (1L PBS+500 .mu.L Tween20)
[0287] Kit Components: [0288] Secondary antibody-PLA probes (plus
and minus) [0289] Ligation reagents [0290] Amplification ligation
reagents [0291] 1.times. Wash Buffer B [0292] 0.01.times. Wash
Buffer B [0293] Mounting media (w/DAPI)
Experimental Procedure:
[0294] 1. Antigen Retrieval: Deparrafinize/Rehydrate slides.
Perform successive washes in
Xylenes and Ethanol Gradient as Follows:
[0295] a. 10 m Xylene
[0296] b. 10 m Xylene (use fresh xylene for this wash, can rotate
forward)
[0297] c. 10 m 50:50 Xylene:Ethanol
[0298] d. 5 m 100% ethanol* Begin setup of pressure cooker here
[0299] e. 5 m 100% ethanol
[0300] f. 5 m 95% ethanol
[0301] g. 5 m 70% ethanol
[0302] h. 5 m 50% ethanol
[0303] i. H2O; can hold here if pressure cooker not ready
[0304] 2. Antigen retrieval (HIER method, using pressure cooker).
Bring 3L of 1.times. PT4 (TE, pH9) [Use a metal slide holder placed
within an eppendorf tube jar] to a low boil in pressure cooker.
Place slide rack in eppendorf tube jar, seal and boil for 20 m.
Remove from heat and let cool for 20 min.
[0305] 3. Wash slide with PBS briefly and use grease pen to trace
along edges of TMA or tissue. Work as quickly as possible
aspirating area around spots in order to delineate area without
spots drying. [0306] a. Record approximate area in cm.sup.2
[0307] 4. Rinse briefly in PB ST (0.05%) and add 1.5% BSA using a
volume sufficient to fully cover delineated area. Ensure that
hydrophopic barrier created by grease pen is maintained. Incubate
30 m RT in humidity chamber.
[0308] 5. Primary antibodies: Add 200-300 .mu.l/slide appropriate
primary antibodies [r-EGFR and m-GRb2] in 0.15% BSA (diluted in
0.05% PBST) Incubate ON in humidity chamber 4.degree. C. with
gentle rocking. [0309] a. Record start time [0310] b. Incubation
time should be 16-18 h
[0311] 6. Next day: Tap off primary antibody solution. Wash 2 times
with sequential immersion in 0.05% PBST in Coplin jars. Wash
2.times.5 m RT with gentle rocking (0.05% PBST).
[0312] 7. PLA probes (secondary antibody-DNA conjugates): Mix and
dilute the two PLA probes 1:5 in 0.15% BSA (diluted in 0.05% PBST)
(use 8 .mu.l minus probe and 8 .mu.l probe plus and 24 .mu.l PBS=40
.mu.l/cm.sup.2). Ensure complete coverage of tissue area. Incubate
slides 1 h 37.degree. C. in pre-heated humidity chamber.
[0313] 8. Tap off PLA probes and wash. Wash 2.times.5 m RT with
gentle rocking (0.05% PBST). Thaw 5.times. ligation mix at
beginning of washes.
[0314] 9. PLA probe ligation: Dilute ligation stock 1:5 in
.sub.MQH.sub.2O while waiting, saving room for ligase (e.g., 8
.mu.l 5.times. ligation mix and 31 .mu.l .sub.MQH.sub.2O=39 .mu.l).
Add Ligase into ligation solution 1:40 during final wash above.
Cover tissue area completely using 40 .mu.l/cm.sup.2. Incubate for
30m 37.degree. C. in pre-warmed humidity chamber.
[0315] 10. Tap off ligation solution and wash. Wash 2.times.2 m RT
with gentle rocking (0.05% PBST). Thaw 5.times. amplification mix
at beginning of washes (avoiding light).
[0316] 11. PLA probe amplification-hybridization-fluorescence:
Dilute Amplification stock 1:5 in .sub.MQH.sub.2O while waiting (8
.mu.l and 31.5 .mu.l .sub.MQH.sub.2O=39 .mu.l). Add Polymerase 1:80
into amplification solution then add polymerase-amplification
solution. Cover tissue area completely using 40 .mu.l/cm.sup.2.
Incubate 2 h 37.degree. C. pre-warmed humidity chamber (avoid
light).
[0317] 12. Tap off amplification solution and rinse slides with
PBS. Incubate with antipan cytokeratin (diluted 1:100) in PBS.
Incubate 1 h 37.degree. C.
[0318] 13. Tap of antibody solution, wash twice with PBS (2.times.5
m RT with gentle rocking).
[0319] 14. Add Alexa-Fluor555, diluted 1:500 in PBS. Incubate 30 m
at 37.degree. C. 15. Tap off aantibody solution and wash 2.times.10
m RT with 1.times. Wash Buffer B. Rinse slide in 0.01.times. Wash
Buffer B.
[0320] 16. Allow slides to dry for .about.10 m in dark. Mount with
Invitrogen Prolong Gold with DAPI. Add mounting media to cover slip
and gently press down onto tissue area. Allow to dry overnight and
image by confocal microscopy next day. Note that Prolong Gold is an
aqueous-based mounting media and will not cure. Care should be
taken to avoid moving coverslip, once mounted. Store slides at
4.degree. after initial overnight dry.
[0321] 17. Confocal Microscopy: Use the Leica TCS SP5 laser
scanning confocal microscope. Place slide on scope and find best
in-focus plane. Take care to avoid imaging along edges of tissue,
whenever possible. Note that Cy5 foci cannot be seen through the
confocal eyepiece due to the far red wavelength. Use 40.times. oil
objective and begin with voltage settings of .about.450 for the 405
laser line (DAPI), .about.600 for the 543 laser line (Cy3) and
.about.580 for the 633 laser line (Cy5). Use 1024.times.1024 image
size and acquire at 200 Hz.
[0322] 18. Use the "live" scan to observe foci. Use the z-plane
wheel to scroll above and below the focus plane until foci
disappear and only DAPI remains, set upper and lower limits.
Generally, 12 z-slices at .76.sub.1.tm provides sufficient coverage
of all foci.
[0323] 19. Do not adjust 633 laser line between samples. DAPI and
Cy3 can be adjusted and will vary among cell types. Keep DAPI
relatively dim as the merged images may be overexposed if DAPI is
too intense.
[0324] 20. After collecting samples (2-3 fields of view for each
sample), process each series as a maximum projection. These images
can then be exported as TIFF files.
EXAMPLE 7
Proximity Ligation Assay for Phosphorylated Rtks in Cell Lines
[0325] Antibodies (varies):
[0326] pEGFRY1068 (Rabbit monoclonal, clone D38B1 Cat# 4267, Cell
Signaling)
[0327] ALK (rabbit monoclonal, clone D5F3 Cat #3633, Cell
Signaling)
[0328] P-Tyr-100 (mouse monoclonal, Cat#9411, Cell Signaling)
[0329] Working dilutions: pEGFRY1068 (1:50), ALK (1:50), P-Tyr-100
(1:1000)
Solutions:
[0330] .sub.MQH.sub.2O
[0331] PBS
[0332] Kit Components: [0333] Secondary antibody-PLA probes (plus
and minus) [0334] Ligation reagents [0335] Amplification ligation
reagents [0336] 1.times. Wash Buffer B [0337] 0.01.times. Wash
Buffer B
[0338] Mounting media (w/DAPI)
Experimental Procedure:
[0339] 1. Plate cells in chamber wells 24 h before processing. For
most cell lines, 50.times.10.sup.4 per well (0.8cm.sup.2) is
sufficient. This number should be adjusted for longer culture times
and different-sized chamber wells.
[0340] 2. Wash each well with PBS (2.times.2 m).
[0341] 3. Fix cells by adding 400 .mu.l 10% Buffered formalin per
well. Incubate 20 m RT with gentle rocking.
[0342] 4. Wash each well with PBS (3.times.2 m).
[0343] 5. Permabilize cells by adding 0.5% Triton X100 (diluted in
PBS). Incubate 10 m RT, no rocking.
[0344] 6. Wash each well with PBS (3.times.2 m).
[0345] 7. Remove chamber from slide. Note that excess silicone
adhesive should be removed with a scalpel to ensure that it does
not interfere with coverslipping later.
[0346] 8. Add 1.5% BSA (diluted in PBS) using a volume sufficient
to fully cover each well. For 8-well slides (0.8 cm.sup.2), a
volume of 50 .mu.l is sufficient. A grease pen can be used to
ensure a hydrophopic barrier exists between wells, but is usually
not necessary. Incubate 30 m RT in humidity chamber.
[0347] 9. Primary antibodies: Add 50 .mu.l per well appropriate
primary antibody soultuion diluted in PBS. Here, can use either a
single antibody or double (depends on epitope availability and
goals of experiment). Incubate ON in humidity chamber 4.degree. C.
with gentle rocking. [0348] a. Record start time [0349] b.
Incubation time should be 16-18 h
[0350] 10. Next day: Tap off primary antibody solution. Wash 2
times with sequential immersion in PBS in Coplin jars. Wash
2.times.5 m RT with gentle rocking.
[0351] 11. PLA probes (secondary antibody-DNA conjugates): Note: if
performing single recognition experiment ensure that a plus and
minus probe from the same species as primary antibody are used. Mix
and dilute the two PLA probes 1:5 (diluted in PBS) (use 8p1 minus
probe and 8 .mu.l probe plus and 24 .mu.l PBS=40 .mu.l/cm.sup.2).
Ensure complete coverage of tissue area. Incubate slides 1 h
37.degree. C. in pre-heated humidity chamber.
[0352] 12. Tap off PLA probes and wash. Wash 2.times.5 m with PBS,
RT with gentle rocking. Thaw 5.times. ligation mix at beginning of
washes.
[0353] 13. PLA probe ligation: Dilute ligation stock 1:5 in
.sub.MQH.sub.2O while waiting, saving room for ligase (e.g., 8
.mu.l 5.times. ligation mix and 31 .mu.l .sub.MQH.sub.2O=39 .mu.l).
Add Ligase into ligation solution 1:40 during final wash above.
Cover tissue area completely using 40 .mu.l/cm.sup.2. Incubate for
30 m 37.degree. C. in pre-warmed humidity chamber.
[0354] 14. Tap off ligation solution and wash. Wash 2.times.2 m RT
with gentle rocking (PBS). Thaw 5.times. amplification mix at
beginning of washes (avoiding light).
[0355] 15. PLA probe amplification-hybridization-fluorescence:
Dilute Amplification stock 1:5 in .sub.MQH.sub.2O while waiting (8
.mu.l and 31.5 .mu.l .sub.MQH.sub.2O=39 .mu.l). Add Polymerase 1:80
into amplification solution then add polymerase-amplification
solution. Cover tissue area completely using 40 .mu.l/cm.sup.2.
Incubate 2 h 37.degree. C. pre-warmed humidity chamber (avoid
light).
[0356] 16. Tap off amplification solution and wash 2.times.10 m RT
with 1.times. Wash Buffer B. Rinse slide in 0.01.times. Wash Buffer
B.
[0357] 17. Allow slides to dry for 10 m in dark. Mount with
Invitrogen Prolong Gold with DAPI. Add mounting media to cover slip
and gently press down onto tissue area. Allow to dry overnight and
image by confocal microscopy next day. Note that Prolong Gold is an
aqueous-based mounting media and will not cure. Care should be
taken to avoid moving coverslip, once mounted. Store slides at
4.degree. after initial overnight dry.
[0358] 18. Confocal Microscopy: Use the Leica TCS SP5 laser
scanning confocal microscope. Place slide on scope and find best
in-focus plane. Take care to avoid imaging along edges of tissue,
whenever possible. Note that Cy5 foci cannot be seen through the
confocal eyepiece due to the far red wavelength. Use 40.times. oil
objective and begin with voltage settings of .about.450 for the 405
laser line (DAPI) and .about.580 for the 633 laser line (Cy5). Use
1024.times.1024 image size and acquire at 200 Hz.
[0359] 19. Use the "live" scan to observe foci. Use the z-plane
wheel to scroll above and below the focus plane until foci
disappear and only DAPI remains, set upper and lower limits.
Generally, 12 z-slices at 0.76 .mu.m provides sufficient coverage
of all foci.
[0360] 20. Do not adjust 633 laser line between samples. DAPI can
be adjusted and will vary among cell types. Keep DAPI relatively
dim as the merged images may be overexposed if DAPI is too
intense.
[0361] 21. After collecting samples (2-3 fields of view for each
sample), process each series as a maximum projection. These images
can then be exported as TIFF files.
EXAMPLE 8
Dulink Assay for Egfr and Grb2 Interaction from Ffpe Tissues With
Brightfield Detection
Reagent Information:
Antibodies:
[0362] GRB2 (Mouse monoclonal, clone 81 (Cat# 610112, BD
Biosciences)
[0363] EGFR (Rabbit monoclonal, clone D38B1 (Cat#4267, Cell
Signaling)
[0364] Working dilutions: m-EGFR (1:100) +r-GRb2 (1:100)
Solutions:
[0365] AR Buffer (PT4) (10 mM Tris Base, 1 mM EDTA, 0.05% Tween 20,
pH9)
[0366] PBS
[0367] .sub.MQH.sub.2O
[0368] 0.05% PBST (1L PBS+500 .mu.L Tween20)
[0369] Kit Components: [0370] Secondary antibody-PLA probes (plus
and minus) [0371] Ligation reagents [0372] Amplification reagents
[0373] Detection reagents [0374] Counterstain reagents [0375]
1.times. Wash Buffer B [0376] 0.01.times. Wash Buffer B [0377]
Mounting media (w/DAPI)
Experimental Procedure:
[0378] 21. Antigen Retrieval: Deparrafinize/Rehydrate slides.
Perform successive washes in
Xylenes and Ethanol Gradient as Follows:
[0379] a. 10 m Xylene [0380] b. 10 m Xylene (use fresh xylene for
this wash, can rotate forward) [0381] c. 10 m 50:50 Xylene:Ethanol
[0382] d. 5 m 100% ethanol* Begin setup of pressure cooker here
[0383] e. 5 m 100% ethanol [0384] f. 5 m 95% ethanol [0385] g. 5 m
70% ethanol [0386] h. 5 m 50% ethanol [0387] i. H2O; can hold here
if pressure cooker not ready
[0388] 22. Antigen retrieval (HIER method, using pressure cooker).
Bring 3L of 1.times. PT4 (TE, pH9) [Use a metal slide holder placed
within an eppendorf tube jar] to a low boil in pressure cooker.
Place slide rack in eppendorf tube jar, seal and boil for 20 m.
Remove from heat and let cool for 20 min.
[0389] 23. Wash slide with PBS briefly and use grease pen to trace
along edges of TMA or tissue. Work as quickly as possible
aspirating area around spots in order to delineate area without
spots drying. [0390] a. Record approximate area in cm.sup.2
[0391] 24. Rinse briefly in PB ST (0/05%) and
[0392] 25. Add 1.5% BSA using a volume sufficient to fully cover
delineated area. Ensure that hydrophopic barrier created by grease
pen is maintained. Incubate 30m RT in humidity chamber.
[0393] 26. Primary antibodies: Add 200-300 .mu.l/slide appropriate
primary antibodies [r-EGFR and m-GRb2] in 0.15% BSA (diluted in
0.05% PBST) Incubate ON in humidity chamber 4.degree. C. with
gentle rocking. [0394] a. Record start time [0395] b. Incubation
time should be 16-18 h
[0396] 27. Next day: Tap off primary antibody solution. Wash 2
times with sequential immersion in 0.05% PBST in Coplin jars. Wash
2.times.5 m RT with gentle rocking (0.05% PB ST).
[0397] 28. PLA probes (secondary antibody-DNA conjugates): Mix and
dilute the two PLA probes 1:5 in 0.15% BSA (diluted in 0.05% PBST)
(use 8 .mu.lminus probe and 8 .mu.l probe plus and 24 .mu.l PBS=40
.mu.l/cm.sup.2). Ensure complete coverage of tissue area. Incubate
slides 1 h 37.degree. C. in pre-heated humidity chamber.
[0398] 29. Tap off PLA probes and wash. Wash 2.times.5 m RT with
gentle rocking (0.05% PBST). Thaw 5.times. ligation mix at
beginning of washes.
[0399] 30. PLA probe ligation: Dilute ligation stock 1:5 in
.sub.MQH.sub.2O while waiting, saving room for ligase (e.g., 8
.mu.l 5.times. ligation mix and 31 .mu.l .sub.MQH.sub.2O=39 .mu.l).
Add Ligase into ligation solution 1:40 during final wash above.
Cover tissue area completely using 40 .mu.l/cm.sup.2. Incubate for
30m 37.degree. C. in pre-warmed humidity chamber.
[0400] 31. Tap off ligation solution and wash. Wash 2.times.2 m RT
with gentle rocking (0.05% PBST). Thaw 5.times. amplification mix
at beginning of washes.
[0401] 32. PLA probe amplification: Dilute Amplification stock 1:5
in .sub.MQH.sub.2O while waiting (8 .mu.l and 31.5 .mu.l
.sub.MQH.sub.2O=39 .mu.l). Add Polymerase 1:80 into amplification
solution then add polymerase-amplification solution. Cover tissue
area completely using 40 .mu.l/cm.sup.2. Incubate 2 h 37.degree. C.
pre-warmed humidity chamber.
[0402] 33. Tap off amplification solution and wash. Wash 2.times.2
m RT with gentle rocking (0.05% PBST). Thaw 5.times. detection mix
at beginning of washes (avoiding light).
[0403] 34. Dilute 5.times. detection reagent in H.sub.2O and add to
slides. Incubate 1 h 37.degree. C.
[0404] 35. Rinse 2.times.5 m in PBST, then add detection reagents
(A, B, C, D) and incubate 10 m.
[0405] 36. Rinse 2.times.2 m in H.sub.2O and add hematoxylin nuclei
stain dropwise. Incubate 2 m RT. Rinse for 10 m under running tap
water.
[0406] 37. Dehydrate slides by incubating in increasing
concentrations of ethanol and then incubate in two changes of
xylene (reversal of step 1 in the protocol). Allow slides to dry in
fume hood.
[0407] 38. Mount slides in toluene-based mounting media and allow
to cure overnight.
[0408] 39. Brightfield Microscopy: Observe slides under light
microscopy at 200.times., 400.times., 630.times. or 1000.times..
Alternatively, slides may be scanned on a slide scanner.
[0409] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application. In addition, any elements
or limitations of any invention or embodiment thereof disclosed
herein can be combined with any and/or all other elements or
limitations (individually or in any combination) or any other
invention or embodiment thereof disclosed herein, and all such
combinations are contemplated with the scope of the invention
without limitation thereto.
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References